Compositions and methods for treatment of disorders related to cep290

ABSTRACT

Compositions are provided that comprise a recombinant vector carrying a nucleic acid sequence encoding a fragment of CEP290 lacking all or part of its N-terminal and C-terminal inhibitory regions, under the control of regulatory sequences which express the product of said gene in a selected cell of a mammalian subject, and a pharmaceutically acceptable carrier. These and other compositions are disclosed with are useful in methods for treating a mammalian subject having a disease associated with a CEP290 mutation, such as Lebers Congenital Amaurosis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No. 14/904,447, filed Jan. 12, 2016, which is a national stage of International Patent application No. PCT/US14/046408, filed Jul. 11, 2014 (expired), which claims the benefit of the priority of U.S. Provisional Patent Application No. 61/847,016, filed Jul. 16, 2013 (expired). The priority applications are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant Nos. R24EY019861 and 8DP1EY023177 awarded by the National Institutes of Health. The government has certain rights in this invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED IN ELECTRONIC FORM

Applicant hereby incorporates by reference the Sequence Listing material filed in electronic form herewith. This file is labeled “UPN-Y6321USD1_ST25.txt”.

BACKGROUND OF THE INVENTION

Defects in primary cilium formation and function are responsible for a variety of human diseases and developmental disorders, collectively termed ciliopathies. While the ciliopathies are diverse in both phenotype and etiology, specific genes, including CEP290, have been implicated as having causative roles in multiple cilium-associated disorders. Mutations in the gene CEP290 have been described in up to 20% of cases of the devastating inherited blinding disease Leber congenital amaurosis and in numerous cases of other more debilitating ciliopathies, such as Joubert syndrome, Senior Loken Syndrome, and Meckel-Gruber syndrome. These disorders range in severity from isolated retinal degeneration to renal dysfunction, central nervous system malformations, hepatic development defects, and embryonic lethality.

Attempts to treat genetic defects have been attempted by delivery of normal genes to the cells expressing the defective genes, such as recombinant adeno-associated virus (AAV)-based therapeutics. See, e.g., U.S. Pat. No. 8,147,823. Generally, large gene sequences have been difficult to fit effectively into some of the more useful vector delivery systems. The extension, to CEP290 patients, of recombinant adeno-associated virus (AAV)-based therapeutics, which have proven safe and effective in the treatment of another genetic cause of LCA, has been hindered by CEP290's large size, precluding it from packaging in AAV.

There remains a need in the art for additional therapeutic compositions and methods for treatment of LCA and other ciliopathies.

SUMMARY OF THE INVENTION

In one aspect, a composition comprises a recombinant vector carrying a nucleic acid sequence encoding a fragment of the CEP290 gene lacking all or portions of at least one of its N-terminal and C-terminal inhibitory regions. The CEP290 fragment is under the control of regulatory sequences which express the product of the gene in a selected cell of a mammalian subject, and a pharmaceutically acceptable carrier. Embodiments comprise nucleic acid sequences of CEP290 which include continuous fragments of CEP290 or discontinuous fragments of CEP290 spliced together in the same reading frame. In one embodiment, the nucleic acid sequence comprises a sequence encoding amino acids 1695 to 1966 (SEQ ID NO: 53) of CEP290, i.e., aa1695 to 1966 of SEQ ID NO: 2. In still another embodiment, the vector of the compositions is an adeno-associated vector.

In another aspect, a composition comprises a recombinant adeno-associated vector carrying a nucleic acid sequence encoding a fragment of the CEP290 gene lacking at least one of its N-terminal and C-terminal inhibitory regions, under the control of regulatory sequences which express the product of said gene in a photoreceptor cell of a mammalian subject, and a pharmaceutically acceptable carrier.

In another aspect, a synthetic or recombinant protein is disclosed that comprises discontinuous CEP290 amino acid fragments spliced together in a single open reading frame. This synthetic protein has biological activity that mimics the biological activity of normal full length CEP290. In certain embodiments, the discontinuous CEP290 amino acid fragments are one or more of aa130 to 380 (SEQ ID NO: 49), aa700 to 1040 (SEQ ID NO: 51), aa1260 to 1605 (SEQ ID NO: 52), aa1695 to 1990 (SEQ ID NO: 54), aa 1695 to 1966 (SEQ ID NO: 53) of CEP290, or aa 1695 to 1995 (SEQ ID NO: 55) of CEP290 of SEQ ID NO: 2 or 4. In another embodiment, the synthetic or recombinant protein has the sequence of SEQ ID NO: 6.

In another aspect, a synthetic or recombinant nucleic acid sequence is provided that encodes the above-referenced protein comprising discontinuous CEP290 amino acid fragments spliced together in a single open reading frame.

In another aspect, compositions containing the synthetic or recombinant protein or nucleic acid sequences also contain therapeutically acceptable carriers. Such compositions may include the vectors above which carry the nucleic acid sequences, or other components.

In yet another aspect, a method of treating a mammalian subject having a disease associated with a CEP290 mutation or defect in the CEP290 gene, protein or expression levels is provided. The method comprises administering to said subject an effective concentration of any of the compositions as described above and in further detail in the specification. This method can involve administering a composition as described herein to cells of the retina, e.g., photoreceptors, the central nervous system, the brain, kidney, bone or olfactory epithelium.

In still another aspect, a method of preventing, arresting progression of or ameliorating vision loss associated with Lebers Congenital Amaurosis in a subject is provided. The method comprises administering to a mammalian subject in need thereof an effective concentration of any of the compositions described herein. In one embodiment, the composition comprises a recombinant adeno-associated virus (AAV) carrying a nucleic acid sequence encoding a fragment of the CEP290 gene lacking at least one of its N-terminal and C-terminal inhibitory regions, under the control of regulatory sequences which express the product of said gene in a photoreceptor cell of a mammalian subject, and a pharmaceutically acceptable carrier.

Other aspects of the invention and disclosure are described in the following detailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A to 1E illustrate that CEP290 aa 1 to 380 (SEQ ID NO: 46), i.e., aa 1 to 380 of SEQ ID NO: 2, mediate membrane association. FIG. 1A is a scale representation of human CEP290 SEQ ID NO: 2. Known human CEP290 mutations are noted by tick marks. The indicated domains and mutations are adapted from reference 30. FIG. 1B are fluorescence microscopy images showing the localization pattern of GFP fusions of full length (FL) and truncated CEP290 constructs in hTERT-RPE1 cells. Cells were stained for ARL13B and with DAPI. Scale bars are 5 μm. Insets show 3× magnified views of areas (in boxes) of colocalization between CEP290 truncations and ARL13B. FIG. 1C show representative membrane flotation assay performed on hTERT-RPE1 cells overexpressing CEP290 aa 1 to 580 (SEQ ID NO: 47) of SEQ ID NO: 2. Equal amounts of each of five fractions, beginning from the top (1) and ending at the bottom (8) of the sucrose gradient were analyzed. The sucrose percentage and the expected protein composition of each fraction are indicated. Blots were probed for GFP to detect CEP290 aa 1 to 580 and for the indicated controls. Relevant molecular weight markers are shown in kDa. FIG. 1D shows that ARL13B positive vesicles were immunoprecipitated from postnuclear supernatants of hTERT-RPE1 cells expressing the indicated CEP290 truncations or GFP alone. The input, unbound fraction (UB) and immunoprecipitated fraction (IP) were probed with an anti-GFP antibody to detect our truncations. Relevant molecular weight markers are shown in kDa. FIG. 1E show the peripheral membrane (Peri.) and integral membrane protein (Int.) fractions of hTERTRPE1 cells expressing the indicated CEP290 truncations that were isolated and probed for GFP to detect CEP290 truncations and for the indicated controls. Relevant molecular weight markers are shown in kDa.

FIGS. 2A to 2E illustrate that CEP290 directly binds membranes in vitro and contains a highly conserved membrane binding amphipathic α-helix. FIG. 2A are fluorescence microscopy images showing the localization pattern of GFP fusions of full length (FL) and truncated CEP290 constructs in hTERTRPE1 cells stained for LAMP2 and with DAPI. Scale bars are 5 μm. Insets show 3× magnified views of areas (in boxes) illustrating lack of colocalization between CEP290 truncations and LAMP2. FIG. 2B shows the results of subcellular fractionation experiments performed on 293T cells expressing CEP290 constructs. Cells were fractionated into cytoplasmic (C), membrane (M), nuclear (N), and cytoskeletal (S) fractions and analyzed by western blotting. Relevant molecular weight markers are shown in kDa. FIG. 2C shows the percent of each truncation present in the membrane fraction. Data are presented as mean±SD, n=3. Asterisks indicate statistical significance over GFP alone. FIG. 2D show the results of liposome co-flotation assays performed on purified CEP290 aa 1 to 580. Data is shown for assays performed with CEP290 aa 1 to 580, both with and without liposomes, and with BSA as a control. Equal amounts of each of five fractions, beginning from the top (1) and ending at the bottom (5) of the sucrose gradient were analyzed. The sucrose percentage and the expected protein composition of each fraction are indicated. Relevant molecular weight markers are shown in kDa. FIG. 2E illustrate helical wheel projection of CEP290 aa 257 to 292 (SEQ ID NO: 50). Darkest circles represent negatively charged amino acids; slightly less dark circles represent positively charged amino acids; the lightest grey circles represent polar, uncharged amino acids, and medium grey circles represent nonpolar amino acids. By aligning CEP290's predicted amphipathic helix from Gallus SEQ ID NO: 35, Meleagris SEQ ID NO: 36, Rattus SEQ ID NO: 37, Mus SEQ ID NO: 38, Pongo SEQ ID NO: 39, Macaca SEQ ID NO: 40, Homo sapiens SEQ ID NO: 41, Felis SEQ ID NO: 42, Ailuropoda SEQ ID NO: 43 and Danio SEQ ID NO: 45, one can see there is usually conservation of polarity and charge between the divergent residues.

FIGS. 3A to 3C show that CEP290 colocalizes with microtubules via region M, and truncation of its N- and C-termini enhances this colocalization. FIG. 3A are fluorescence confocal microscopy images showing the localization pattern of GFP fusions of full length (FL) and truncated CEP290 constructs expressed in hTERT-RPE1 cells or in HEK293T cells. Samples were stained for α-tubulin or acetylated α-tubulin, pericentrin or with DAPI. Scale bars are 10 μm. Insets show 10× magnified views of areas of colocalization (in boxes) between CEP290 truncations and pericentrin. In all cases, truncations partially colocalize with pericentrin. In some cases, truncations colocalize with tubulin. Region M is necessary and sufficient for tubulin colocalization. FIG. 3B are scale representations of the library of CEP290 truncations tested in FIG. 3A. Included is a summary of the extent of colocalization with tubulin, rated on a scale ranging from negative (−) to highly positive. Truncations were also in vitro transcribed and translated and subjected to a microtubule MT sedimentation assay. The recombinantly expressed and purified microtubule binding region of CEP290 was similarly subjected to a MT sedimentation assay. FIG. 3C illustrates that for each truncation, the percent of transfected hTERT-RPE1 cells in which the GFP fused truncation displayed a fibrillar localization pattern was determined. At least 100 transfected cells were counted per experiment. Data are presented as mean±SD, n=3. Quantification of the different localization patterns of each truncation from three independent experiments. Region M of CEP290 is necessary and sufficient for fibrillar localization. The inclusion of the N- and C-termini diminishes the extent to which truncations assume a fibrillar pattern of localization.

FIGS. 4A to 4F show that CEP290 directly binds to and bundles microtubules via region M, and its N- and C-termini inhibit microtubule binding and bundling. FIG. 4A shows representative confocal fluorescence microscopy images of GFP fusions of full length (FL) and truncated CEP290 constructs expressed in hTERT-RPE1 cells stained for acetylated α-tubulin and with DAPI. Scale bars are 10 μm. Truncations containing CEP290 aa1695 to 1966 of SEQ ID NO: 2 perturb the acetylated tubulin staining pattern in hTert-RPI and 293T cells, and deletion of the N- and C-termini enhance the effect. Confocal images of CEP290 truncations overexpressed in hTERT-RPE1 cells stained with an anti-acetylated tubulin antibody. Truncations containing aa1695 to 1966 of SEQ ID NO: 2 increase the intensity of acetylated tubulin staining and result in more bundles of acetylated MTs compared to untransfected cells. FIG. 4B show percent of transfected 293T cells showing an increase in the intensity, and perturbation in pattern, of acetylated α-tubulin staining compared to untransfected cells, following transfection with CEP290 truncations. Deletion of both the N- and C-termini, but not either alone, significantly increases the intensity of acetylated tubulin staining. 100 transfected cells were counted per experiment. Data are presented as mean±SD, n=5. Means with different letters are significantly different. FIG. 4C are Western blots of microtubule (MT) co-sedimentation assays for in vitro transcribed and translated CEP290 truncation mutants. The resulting supernatants (S) and microtubule pellets (P) were probed for the presence of the GFP-fused truncations and are shown for assays performed both with (+MT) and without (−MT) microtubules. Relevant molecular weight markers are shown in kDa. FIG. 4D shows the percent of each truncation co-sedimenting with microtubules. Quantification of the percent of total truncation present in the MT pellet for three independent experiments is shown. Only minimal pelleting is observed in the −MT controls. AA1695 to 1966 of SEQ ID NO: 2 are necessary for significant MT binding, and deletion of both the N- and C-termini, but not of either alone, significantly increases MT binding. Data are presented as mean±SD, n=3. Means with different letters are significantly different. The N- and C-termini cooperatively inhibit MT binding. FIG. 4E are Coomassie-stained gel of microtubule co-sedimentation assays performed with recombinantly expressed and purified CEP290 truncation M subjected to a MT sedimentation assay with increasing concentrations of microtubules (tubulin). Truncation M directly binds MTs with an apparent K_(d) in the nanomolar range, comparable to other MT binding proteins. The supernatants (S) and microtubule pellets (P) are shown. Relevant molecular weight markers are shown in kDa. CEP290 aa1695 to 1966 of SEQ ID NO: 2 directly bind MTs with a K_(d) in the nanomolar range. FIG. 4F is a binding curve of microtubule cosedimentation assays as in FIG. 4E. The fraction of truncation M present in the pellet in the absence of microtubules was subtracted from all data points. Data points are presented as means±SD, n=3. A curve was fit to the data points using the non-linear regression functionality of the GraphPad Prism program.

FIGS. 5A to 5G show that the overexpression of either the N- or C-terminal regulatory regions of CEP290 ablates the normal inhibition of CEP290. FIG. 5A shows fluorescence microscopy fields of hTERT-RPE1 cells transduced with lentiviral empty vector (EV), or vectors encoding either the N- (aa 1 to 580) or C-terminus (aa 1966 to 2479) of CEP290 (SEQ ID NO: 59). Cells were stained for acetylated α-tubulin and pericentrin to detect primary cilia and with DAPI. Arrowheads indicate primary cilia. Arrows indicate cells with multiple axonemes originating from the same focus of pericentrin. Scale bars are 10 μm. FIG. 5B show the percent of lentivirus-treated cells forming primary cilia. Data are presented as mean±SD, n=3. 100 cells were counted per experiment. FIG. 5C show fluorescence microscopy images of single hTERT-RPE1 cells transduced with lentiviral vectors as in FIG. 5A. Cells were stained for acetylated α-tubulin to detect primary cilia and with DAPI. Scale bars are 5 μm. FIG. 5D shows the average primary cilium length for hTERT-RPE1 cells as in FIG. 5C. Data are presented as mean±SD, n=3. A total of at least 150 cilia were measured per condition. FIG. 5E are fluorescence microscopy images of hTERT-RPE1 cells that formed multiple cilia after transduction with lentiviral vector encoding the N-terminus of CEP290. Cells were stained with acetylated α-tubulin, ARL13B, and with DAPI. Scale bars are 5 μm. FIG. 5F are fluorescence microscopy images of hTERT-RPE1 cells that formed multiple cilia after transduction with lentiviral vector encoding the N-terminus of CEP290. Cells were stained with acetylated α-tubulin, pericentrin and with DAPI. Scale bars are 5 μm. FIG. 5G shows the percent of lentivirus treated hTERT-RPE1 cells forming multiple cilia. At least 100 cells were counted per experiment. Data are presented as mean±SD, n=3.

FIGS. 6A to 6F show that an in-frame deletion in Cep290 in the rd16 mouse ablates Cep290's microtubule binding activity. FIG. 6A is a schematic representation of the microtubule binding region of human CEP290 in relation to the rd16 mouse deletion (17). Truncations of CEP290 representing the part of the microtubule binding region deleted in the rd16 mouse and the part of the microtubule binding region maintained in the rd16 mouse were created as shown. FIG. 6B are confocal fluorescence microscopy images showing the localization pattern of the GFP-tagged “Maintained” and “Deleted” CEP290 truncations. Cells were stained for α-tubulin, pericentrin and with DAPI. Scale bars are 10 μm. FIG. 6C are immunoblots of representative microtubule co-sedimentation assays for in vitro transcribed and translated “Maintained” and “Deleted” CEP290 truncations. The supernatant (S) and microtubule pellet (P) fractions are shown in assays performed both with (+MT) and without (−MT) the addition of microtubules. Relevant molecular weight markers are shown in kDa. FIG. 6D shows the percent of each truncation co-sedimenting with microtubules. Data are presented as mean±SD, n=2. FIG. 6E are representative western blots of microtubule co-sedimentation assays performed with full length WT and rd16 Cep290 from mouse brain homogenate. The supernatant (S) and microtubule pellet (P) fractions are shown in assays performed on samples induced to polymerize microtubules (+MT) and samples treated to prevent microtubule polymerization (−MT). Relevant molecular weight markers are shown in kDa. FIG. 6F shows percent of WT and rd16 CEP290 co-sedimenting with microtubules. Data are presented as mean±SD, n=3. Asterisks indicate statistical significance over −MT samples.

FIG. 7A to 7E show rd16 mouse fibroblasts are deficient in primary cilium formation. FIG. 7A show fluorescence microscopy fields of WT primary dermal fibroblasts that were stained for acetylated α-tubulin to identify primary cilia, and stained with DAPI. Fibroblasts were grown in media with (Fed) or without (Starved) serum. Arrowheads indicate primary cilia. Scale bars are 10 μm. FIG. 7B show fluorescence microscopy fields of rd16 primary dermal fibroblasts stained for acetylated α-tubulin to identify primary cilia, and stained with DAPI. Fibroblasts were grown in media with (Fed) or without (Starved) serum. Arrowheads indicate primary cilia. Scale bars are 10 μm. FIG. 7C show the percent of WT and rd16 primary dermal fibroblasts that form primary cilia in serum fed and serum starved conditions. Quantification was based on separate experiments on fibroblasts coming from 3 different animals per genotype. At least 100 cells were counted per experiment. Data are presented as mean±SD, n=5. FIG. 7D show high magnification fluorescent microscopy images of representative serum-starved WT and rd16 primary dermal fibroblasts stained for acetylated α-tubulin to identify primary cilia, and stained with DAPI. Scale bars are 5 μm. FIG. 7E are average cilium length of serum starved WT and rd16 primary dermal fibroblasts. Quantification was based on separate experiments on fibroblasts coming from 3 different animals per genotype. At least 50 cilia were measured per experiment, and a total of 400 cilia were measured for both the WT and rd16 fibroblasts. Data are presented as mean±SD, n=5.

FIG. 8 shows a speculative model for CEP290 activity at the primary cilium in four panels. CEP290 is maintained in a closed, inhibited state by its N- and C-termini and CP110 during the cell cycle; CP110 bound to CEP290 with the N- and C-termini binding each other (panel A). Upon entry into G₀ CP110 is degraded at the mother centriole (panel B), destabilizing the closed conformation. This conformational change in the protein frees and activates CEP290's membrane binding and microtubule binding domains (panel C). In its open conformation, active CEP290 is able to recruit additional interacting partners including MTs and to initiate IFT and ciliogenesis (panel D).

FIG. 9A is the nucleic acid sequence encoding full length naturally occurring human CEP290 SEQ ID NO: 1.

FIG. 9B is the amino acid sequence encoding full length naturally occurring human CEP290 SEQ ID NO: 2.

FIG. 10A is a synthetic nucleic acid sequence for codon optimized human CEP290 SEQ ID NO: 3.

FIG. 10B is a synthetic amino acid sequence for codon optimized human CEP290 SEQ ID NO: 4.

FIG. 11A is a synthetic construct which is a minigene for CEP290 SEQ ID NO: 5, which encodes the following CEP290 amino acid fragments, spliced together in a single open reading frame: aa130 to 380, aa700 to 1040, aa1260 to 1605, and aa1695 to 1990.

FIG. 11B is the synthetic amino acid sequence construct SEQ ID NO: 6 encoded by FIG. 11A.

FIGS. 12A to 12C demonstrate the generation and testing of CEP290 shRNA constructs. FIG. 12A shows hTERT-RPE1 cells transiently transfected with three different CEP290 shRNA constructs, which were lysed and analyzed by western blotting for CEP290 levels. Blots were reprobed for GAPDH as a loading control. FIG. 12B shows densitometric quantification of CEP290 protein levels in hTERT-RPE1 cells as in FIG. 12A. CEP290 levels were normalized using GAPDH as a loading control and the percent of CEP290 remaining, compared to untransfected hTERT-RPE1 cells, was determined. Immunofluorescence microscopy images of fields of hTERT-RPE1 cells, either untransfected or transfected with CEP290 shRNA construct 2, were stained for acetylated tubulin as a marker of the primary cilium (data not shown). FIG. 12C shows the percent of control and sh2 transfected hTERT-RPE1 cells that formed cilia upon serum starvation.

FIGS. 13A to 13C show the isolation and testing of clonal CEP290 knockdown cell lines. FIG. 13A shows clonal retrovirus-transduced hTERT-RPE1 cell lines expressing CEP290 shRNA 2 that were lysed and analyzed by western blotting for CEP290 levels. Blots were reprobed for GAPDH as a loading control. FIG. 13 B shows densitometric quantification of CEP290 protein levels in these hTERT-RPE1 cells. CEP290 levels were normalized using GAPDH as a loading control and the percent of CEP290 remaining, compared to untransfected hTERT-RPE1 cells, was determined. Immunofluorescence microscopy images of fields of control and sh2.8 hTERT-RPE1 cells stained for acetylated tubulin as a marker of the primary cilium were obtained (data not shown). FIG. 13C shows the percent of control and sh2.8 hTERT-RPE1 cells that formed cilia upon serum starvation.

FIGS. 14A and 14B show the construction and testing of a miniCEP290 construct. FIG. 14A is a schematic representation of the CEP290 protein with identified functional domains, interacting domains, and protein motifs labeled. Grayed areas represent regions of the protein not included in the miniCEP290 construct. FIG. 14B is a schematic representation of miniCEP290. Immunofluorescence images of hTERT-RPE1 cells transiently transfected with GFP-fused miniCEP290 and stained for acetylated tubulin and pericentrin, and with DAPI were obtained, and showed the localization of miniCEP290 to the ciliary transition zone (data not shown).

DETAILED DESCRIPTION OF THE INVENTION

CEP290 is a vital structural and regulatory element of the ciliary transition zone, elucidation of its molecular functionality at the center of the critically important and disease-relevant pathways of ciliogenesis and IFT. The inventors have identified novel regulatory domains of CEP290 useful in therapeutic interventions for diseases of the cilia caused by mutated CEP290. Truncation mutants or fragments of CEP290 lacking certain of the novel inhibitory domains but maintaining the other functional regions of the protein exhibit normal, or even enhanced, CEP290 function, while at the same time being small enough to fit in certain vectors, such as AAV. The delivery of such a therapeutic to terminally differentiated tissues, such as the retina, to effect permanent activation of CEP290 is useful in the treatment of CEP290-related diseases, such as LCA. The delivery of such a therapeutic is also useful to treat other diseases caused by naturally mutated or non-functional CEP290 in other tissues, such as the brain or kidney or bone.

Four novel functional domains of the CEP290 protein are identified, showing that CEP290 directly binds to the cellular membrane via an N-terminal domain that includes a highly conserved amphipathic helix motif, and directly binds to microtubules through a domain located within its myosin-tail homology domain. Furthermore, CEP290 activity was found to be regulated by two novel autoinhibitory domains within its N- and C-termini, both of which were also found to play critical roles in regulating ciliogenesis. Disruption of the novel microtubule binding domain in the rd16 mouse LCA model was found to be sufficient to induce significant deficits in cilium formation leading to retinal degeneration. Various compositions and treatment methods for CEP290-related diseases utilizing these domains are disclosed.

In one embodiment, a composition comprises a recombinant vector carrying a nucleic acid sequence encoding a fragment of the CEP290 gene lacking at least one of its N-terminal and C-terminal inhibitory regions, under the control of regulatory sequences which express the product of said gene in a selected cell of a mammalian subject, and a pharmaceutically acceptable carrier. In another embodiment, such a composition comprises an effective concentration of a recombinant adeno-associated virus (rAAV) carrying a nucleic acid sequence encoding a CEP290 fragment or truncated gene, as described herein, under the control of regulatory sequences which direct expression of the product of the gene in the subject's ocular cells, formulated with a carrier and additional components suitable for injection. In still another embodiment, the treatment methods are directed to ocular disorders and associated conditions related thereto. Other treatment methods are also disclosed.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and by reference to published texts, which provide one skilled in the art with a general guide to many of the terms used in the present application. The definitions used herein are provided for clarity only and are not intended to limit the claimed invention.

As used herein, the term “mammalian subject” or “subject” includes any mammal in need of these methods of treatment or prophylaxis, including particularly humans. Other mammals in need of such treatment or prophylaxis include dogs, cats, or other domesticated animals, horses, livestock, laboratory animals, including non-human primates, etc. The subject may be male or female. In another embodiment, the subject has shown clinical signs of ciliopathy, such as Lebers Congenital Amaurosis. Clinical signs of LCA include, but are not limited to, decreased peripheral vision, decreased central (reading) vision, decreased night vision, loss of color perception, reduction in visual acuity, decreased photoreceptor function, pigmentary changes. In another embodiment, the subject has been diagnosed with LCA. In yet another embodiment, the subject has not yet shown clinical signs of LCA. In still another embodiment, the subject has shown signs or symptoms of another ciliopathy, e.g., disorders of the bone, brain, CNS, kidney, or olfactory epithelia.

As used herein, the term “selected cells” refers to any cell in which a CEP290 mutation causes disease. In one embodiment, the selected cell is an ocular cell, which is any cell associated with the function of, the eye. In one embodiment, the ocular cell is a photoreceptor cells. In another embodiment, the term refers to rod, cone and photosensitive ganglion cells or retinal pigment epithelium (RPE) cells. In another embodiment, the selected cell is a bone cell. In another embodiment, the selected cell is a brain cell or neuron. In another embodiment, the selected cell is a renal or kidney cell. In another embodiment, the selected cell is a mucosal cell, such as an olfactory epithelial cell.

“CEP290 related pathologies” or those caused by a defect or mutation in CEP290 include Leber congenital amaurosis, Joubert syndrome, Senior Loken Syndrome, and Meckel-Grüber syndrome, including isolated retinal degeneration, renal dysfunction, central nervous system malformations, hepatic development defects, and embryonic lethality.

The terms “a” or “an” refers to one or more, for example, “a gene” is understood to represent one or more such genes. As such, the terms “a” (or “an”), “one or more,” and “at least one” are used interchangeably herein. As used herein, the term “about” means a variability of 10% from the reference given, unless otherwise specified.

With regard to the following description, it is intended that each of the compositions herein described, is useful, in another embodiment, in the methods of the invention. In addition, it is also intended that each of the compositions herein described as useful in the methods, is, in another embodiment, itself an embodiment of the invention. While various embodiments in the specification are presented using “comprising” language, under other circumstances, a related embodiment is also intended to be interpreted and described using “consisting of” or “consisting essentially of” language.

CEP290 Nucleic Acid and Proteins and Fragments Thereof

“CEP290” is a mammalian gene of about 7.8 kb in size (see NCBI database ref gene 80184 for the full length human gene sequence) which encodes a full length protein of about 2479 amino acids (See e.g., SEQ ID NO: 1 for the human gene sequence). CEP290 participates in the formation of the primary cilium (an organelle found in nearly all cell types) and regulates the trafficking of proteins into and out of the ciliary compartment. Mutations in CEP290 lead to aberrant ciliary trafficking, eventually resulting in pathologies. Mutations in this gene have been associated with a variety of disease functions. It is implicated in LCA, as well as other ciliopathies affecting numerous organ systems, including the retina, CNS, kidney, liver heart and bone.

The nucleic acid sequence encoding a normal CEP290 gene may be derived from any mammal which natively expresses the CEP290 gene, or homolog thereof. In another embodiment, the CEP290 gene sequence is derived from the same mammal that the composition is intended to treat. In another embodiment, the CEP290 is derived from a human. In other embodiments, certain modifications are made to the CEP290 sequence in order to enhance the expression in the target cell. Such modifications include codon optimization. Codon optimization may be performed in a manner such as that described in, e.g., U.S. Pat. Nos. 7,561,972; 7,561,973; and 7,888,112, incorporated herein by reference, and conversion of the sequence surrounding the translational start site to a consensus Kozak sequence. See, Kozak et al, Nucleic Acids Res. 15 (20): 8125-8148, incorporated herein by reference.

A full length human nucleic acid sequence for CEP290 is shown as SEQ ID NO. 1 and its full length protein sequence is shown as SEQ ID NO: 2. A full length codon-optimized version of a human nucleic acid sequence is identified herein as SEQ ID NO: 3 with its full length protein sequence identified as SEQ ID NO:4. Throughout this disclosure, numbering of the amino acid fragments of CEP is that of the amino acid sequence of FIG. 10B, SEQ ID NO:2 with the first amino acid of FIG. 10B numbered as 1.

A “CEP290 fragment or truncation” as used herein is defined as a fragment of CEP290 lacking the sequences that inhibit the protein's function. By the term “fragment” or “functional fragment” is meant any fragment that retains the function of the full length CEP290, although not necessarily at the same level of expression or activity. As disclosed herein CEP290 is a microtubule binding protein, with its MT binding activity localized to aa 1695 to 1966 of SEQ ID NO: 2, i.e., the sequence

DQSQKESQCLKSELQAQKEANSRAPTTTMRNLVERLKSQLALKEKQQKAL SRALLELRAEMTAAAEERIISATSQKEAHLNVQQIVDRHTRELKTQVEDL NENLLKLKEALKTSKNRENSLTDNLNDLNNELQKKQKAYNKILREKEEID QENDELKRQIKRLTSGLQGKPLTDNKQSLIEELQRKKKLENQLEGKVEEV DLKPMKEKNAKEELIRWEEGKKWQAKIEGIRNKLKEKEGEVFTLTKQLNT LKDLFAKADKEKLTLQRKLKT.

The inventors identified inhibitory regions at the N and C termini thereof. The inventors have also determined that the N- and C termini of the protein cooperatively inhibit the MT binding activity. As disclosed in detail in the examples below, the inventors determined that the nucleic acid sequence of CEP290 encoding amino acids spanning aa1695 to 1966 were necessary for colocalization with microtubules. While inhibitory functions were found within the N terminal sequences encoding aa 1 to 580, other sequences located at the N-terminus were functional in other ways, e.g., the sequences encoding amino acids 1 to 380 were determined to contain regions necessary for vesicular localization and amino acids 1 to 362 (SEQ ID NO: 45) were determined to contain regions necessary for membrane association. Amino acids 257 to 292 were found to be the alpha helix and necessary for reversible interaction. The C-terminal portions of the protein, from about amino acid 2000 to 2479 (SEQ ID NO: 58) or about 1967 to 2479 (SEQ ID NO: 57), were found to cooperatively inhibit microtubule binding with portions of the N-terminal inhibitory sequence within amino acids 1 to 580.

CEP290 nucleic acid fragments or truncated sequences for use in the therapeutic methods described herein can be a single consecutive sequence, such as that encoding CEP290 protein lacking its N terminal membrane binding inhibitory region. In another embodiment, the nucleic acid sequence fragment encodes CEP290 lacking its C terminal membrane binding inhibitory region. In still another useful embodiment, the nucleic acid sequence fragment encodes CEP290 lacking both the N-terminal and C-terminal inhibitory regions. CEP290 fragments consist of a single consecutive CEP290 sequence or spliced together fragments of one or more fragments of CEP290, which together provide the regions necessary for protein function. Such fragments can individually be from about 150 to over 1000 nucleotides in length. The encoded fragments can be from about 50 to over 300 amino acids in length.

Thus in one embodiment a useful CEP209 nucleic acid sequence encodes aa1695 to about 2000 of CEP290 (SEQ ID NO: 56). In one embodiment, the nucleic acid sequence fragment encodes aa 1695 to aa 1990. In another embodiment, the sequence encodes aa 1695 to 1966. Still other nucleic acid sequences encoding continuous or discontinuous amino acids sequences within that range can be included as useful fragments herein. In another embodiment, a useful CEP290 fragment encodes aa100 to 362 (SEQ ID NO: 48). In another embodiment the useful CEP290 fragment encodes amino acids 130 to 380 of CEP290. In another embodiment the useful CEP290 fragment encodes amino acids 130 to 380 of CEP290. In another embodiment the useful CEP290 fragment encodes amino acids 700 to 1040 of CEP290. In another embodiment the useful CEP290 fragment encodes amino acids 1260 to 1605 of CEP290. In still another embodiment, spliced together fragments of one or more of the fragments of CEP290 disclosed herein provide the regions necessary for protein function. In another embodiment, to form a useful CEP290 fragment, the nucleic acid sequences of CEP290 are spliced together in the same reading frame. In one embodiment, the CEP290 fragment is useful for delivery to a mammalian cell to replace a mutated version of CEP290 is a nucleic acid minigene encoding CEP fragments spliced together in a single reading frame: aa130 to 380, aa 700 to 1040, aa 1260 to 1605, and aa1695 to 1990. One exemplary CEP290 minigene sequence is shown as SEQ ID NO: 5, with is encoded sequence being SEQ ID NO: 6.

In still another embodiment, the fragment of CEP290 is derived from nucleic acid sequence SEQ ID NO: 1. In another embodiment, the fragment of CEP290 is derived from human codon-optimized nucleic acid sequence SEQ ID NO: 3. In still another embodiment the nucleic acid sequence encoding a fragment of CEP290 is derived from or is the minigene sequence SEQ ID NO: 5. In one embodiment, the CEP290 fragment is useful for delivery to a mammalian cell to replace a mutated version.

It is anticipated that the CEP290 nucleic acid fragments and the CEP290 protein truncates or amino acid fragments identified herein may tolerate certain minor modifications at the nucleic acid level to include, for example, modifications to the nucleotide bases which are silent, e.g., preference codons. In other embodiments, nucleic acid base modifications which change the amino acids, e.g. to improve expression of the resulting peptide/protein are anticipated. Also included as likely modification of fragments are allelic variations, caused by the natural degeneracy of the genetic code. Also included as modification of the CEP290 expressed fragments are analogs, or modified versions, of the CEP290 protein fragments provided herein. Typically, such analogs differ from the specifically identified CEP290 proteins by only one to four codon changes. Examples include CEP290 fragments with conservative amino acid replacements from the CEP290 sequence from which the fragment is derived. Conservative replacements are those that take place within a family of amino acids that are related in their side chains and chemical properties. CEP290 fragments which have at least 80% sequence identity with the above derived fragments from known sequences of CEP290, including the SEQ ID NOs: 1 to 4 herein, are anticipated to be useful in the compositions and methods of this invention.

The Vectors

A variety of known nucleic acid vectors may be used in these methods, e.g., recombinant viruses, such as recombinant adeno-associated virus (AAV), recombinant adenoviruses, recombinant retroviruses, recombinant poxviruses, and other known viruses in the art, as well as plasmids, cosmids and phages, etc. A wealth of publications known to those of skill in the art discusses the use of a variety of such vectors for delivery of genes (see, e.g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989; Kay, M. A. et al, 2001 Nat. Medic., 7(1):33 to 40; and Walther W. and Stein U., 2000 Drugs, 60(2):249 to 71). In one embodiment of this invention the vector is a recombinant AAV carrying a CEP290 fragment cDNA driven by a promoter that expresses the product of the CEP290 nucleic acid sequence in selected cells of the affected subject. Methods for assembly of the recombinant vectors are well-known (see, e.g., International Patent Publication No. WO 00/15822, published Mar. 23, 2000 and other references cited herein). To exemplify the methods and compositions of this invention, the presently preferred vector, a recombinant AAV is described in detail.

In certain embodiments of this invention, the CEP290 nucleic acid sequence, or fragment thereof, is delivered to the selected cells, e.g., photoreceptor cells, in need of treatment by means of a viral vector, of which many are known and available in the art. In certain embodiments, the therapeutic vector is desirably non-toxic, non-immunogenic, easy to produce, and efficient in protecting and delivering DNA into the target cells. In one particular embodiment, the viral vector is an adeno-associated virus vector.

More than 30 naturally occurring serotypes of AAV are available. Many natural variants in the AAV capsid exist, allowing identification and use of an AAV with properties specifically suited for ocular cells. AAV viruses may be engineered by conventional molecular biology techniques, making it possible to optimize these particles for cell specific delivery of RPGR nucleic acid sequences, for minimizing immunogenicity, for tuning stability and particle lifetime, for efficient degradation, for accurate delivery to the nucleus, etc.

The expression of CEP290 functional nucleic acid fragments can be achieved in the selected cells through delivery by recombinantly engineered AAVs or artificial AAV's that contain sequences encoding the desired CEP290 fragment. The use of AAVs is a common mode of exogenous delivery of DNA as it is relatively non-toxic, provides efficient gene transfer, and can be easily optimized for specific purposes. Among the serotypes of AAVs isolated from human or non-human primates (NHP) and well characterized, human serotype 2 is the first AAV that was developed as a gene transfer vector; it has been widely used for efficient gene transfer experiments in different target tissues and animal models. Clinical trials of the experimental application of AAV2 based vectors to some human disease models are in progress, and include such diseases as cystic fibrosis and hemophilia B. Other AAV serotypes include, but are not limited to, AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 and AAV9. See, e.g., WO 2005/033321 for a discussion of various AAV serotypes, which is incorporated herein by reference. Still other modified AAV8 sequences are described in U.S. patent application No. 61/762,775, filed Feb. 8, 2013, incorporated by reference herein. For use in photoreceptor cells, a modified AAV 8_b is a useful vector, among others.

Desirable AAV fragments for assembly into vectors include the cap proteins, including the vp1, vp2, vp3 and hypervariable regions, the rep proteins, including rep 78, rep 68, rep 52, and rep 40, and the sequences encoding these proteins. These fragments may be readily utilized in a variety of vector systems and host cells. Such fragments may be used alone, in combination with other AAV serotype sequences or fragments, or in combination with elements from other AAV or non-AAV viral sequences. As used herein, artificial AAV serotypes include, without limitation, AAV with a non-naturally occurring capsid protein. Such an artificial capsid may be generated by any suitable technique, using a selected AAV sequence (e.g., a fragment of a vp1 capsid protein) in combination with heterologous sequences which may be obtained from a different selected AAV serotype, non-contiguous portions of the same AAV serotype, from a non-AAV viral source, or from a non-viral source. An artificial AAV serotype may be, without limitation, a pseudotyped AAV, a chimeric AAV capsid, a recombinant AAV capsid, or a “humanized” AAV capsid. Pseudotyped vectors, wherein the capsid of one AAV is replaced with a heterologous capsid protein, are useful in the invention. In one embodiment, AAV2/5 a useful pseudotyped vector. In another preferred embodiment, the AAV is AAV2/8. See, Mussolino et al, cited above.

In one embodiment, the vectors useful in compositions and methods described herein contain, at a minimum, sequences encoding a selected AAV serotype capsid, e.g., an AAV8 capsid, or a fragment thereof. In another embodiment, useful vectors contain, at a minimum, sequences encoding a selected AAV serotype rep protein, e.g., AAV8 rep protein, or a fragment thereof. Optionally, such vectors may contain both AAV cap and rep proteins. In vectors in which both AAV rep and cap are provided, the AAV rep and AAV cap sequences can both be of one serotype origin, e.g., all AAV8 origin. Alternatively, vectors may be used in which the rep sequences are from an AAV serotype which differs from that which is providing the cap sequences. In one embodiment, the rep and cap sequences are expressed from separate sources (e.g., separate vectors, or a host cell and a vector). In another embodiment, these rep sequences are fused in frame to cap sequences of a different AAV serotype to form a chimeric AAV vector, such as AAV2/8 described in U.S. Pat. No. 7,282,199, which is incorporated by reference herein.

A suitable recombinant adeno-associated virus (AAV) is generated by culturing a host cell which contains a nucleic acid sequence encoding an adeno-associated virus (AAV) serotype capsid protein, or fragment thereof, as defined herein; a functional rep gene; a minigene composed of, at a minimum, AAV inverted terminal repeats (ITRs) and a RPGR nucleic acid sequence; and sufficient helper functions to permit packaging of the minigene into the AAV capsid protein. The components required to be cultured in the host cell to package an AAV minigene in an AAV capsid may be provided to the host cell in trans. Alternatively, any one or more of the required components (e.g., minigene, rep sequences, cap sequences, and/or helper functions) may be provided by a stable host cell which has been engineered to contain one or more of the required components using methods known to those of skill in the art.

Most suitably, such a stable host cell will contain the required component(s) under the control of an inducible promoter. The required component(s) may be under the control of a constitutive promoter. The promoter of the host cell can also be the desired promoter for the vector used in the therapeutic compositions, and may depend upon the selected cell which is ultimately to be treated. Examples of suitable inducible and constitutive or cell/tissue specific promoters are provided herein, in the discussion below of regulatory elements suitable for use with the transgene, i.e., a functional fragment of CEP290. In still another alternative, a selected stable host cell may contain selected component(s) under the control of a constitutive promoter and other selected component(s) under the control of one or more inducible promoters. For example, a stable host cell may be generated which is derived from 293 cells (which contain E1 helper functions under the control of a constitutive promoter), but which contains the rep and/or cap proteins under the control of inducible promoters. Still other stable host cells may be generated by one of skill in the art.

The minigene, rep sequences, cap sequences, and helper functions required for producing the rAAV of the invention may be delivered to the packaging host cell in the form of any genetic element which transfers the sequences carried thereon. The selected genetic element may be delivered by any suitable method, including those described herein. The methods used to construct any embodiment of this invention are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. Similarly, methods of generating rAAV virions are well known and the selection of a suitable method is not a limitation on the present invention. See, e.g., K. Fisher et al, 1993 J. Virol., 70:520 to 532 and U.S. Pat. No. 5,478,745, among others. These publications are incorporated by reference herein.

Unless otherwise specified, the AAV ITRs, and other selected AAV components described herein, may be readily selected from among any AAV serotype, including, without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or other known and unknown AAV serotypes. These ITRs or other AAV components may be readily isolated using techniques available to those of skill in the art from an AAV serotype. Such AAV may be isolated or obtained from academic, commercial, or public sources (e.g., the American Type Culture Collection, Manassas, Va.). Alternatively, the AAV sequences may be obtained through synthetic or other suitable means by reference to published sequences such as are available in the literature or in databases such as, e.g., GenBank, PubMed, or the like.

The desired AAV minigene is composed of, at a minimum, a CEP290 functional fragment nucleic acid sequence (the transgene, e.g., such as the fragment of SEQ ID NO: 5), as described herein, and its regulatory sequences, and 5′ and 3′ AAV inverted terminal repeats (ITRs). In one embodiment, the ITRs of AAV serotype 2 are used. In another embodiment, the ITRs of AAV serotype 5 or 8 are used. However, ITRs from other suitable serotypes may be selected. It is this minigene which is packaged into a capsid protein and delivered to a selected host cell.

The Regulatory Sequences

The regulatory sequences include conventional control elements which are operably linked to the CEP290 gene fragment or minigene in a manner which permits its transcription, translation and/or expression in a selected cell transfected with the vector or infected with the virus produced by the invention. As used herein, “operably linked” sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.

Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. A great number of expression control sequences, including promoters, are known in the art and may be utilized.

The regulatory sequences useful in the constructs of the present invention may also contain an intron, desirably located between the promoter/enhancer sequence and the gene. One desirable intron sequence is derived from SV to 40, and is a 100 bp mini-intron splice donor/splice acceptor referred to as SD-SA. Another suitable sequence includes the woodchuck hepatitis virus post-transcriptional element. (See, e.g., L. Wang and I. Verma, 1999 Proc. Natl. Acad. Sci., USA, 96:3906 to 3910). PolyA signals may be derived from many suitable species, including, without limitation SV-40, human and bovine.

Another regulatory component of the rAAV useful in the method of the invention is an internal ribosome entry site (IRES). An IRES sequence, or other suitable systems, may be used to produce more than one polypeptide from a single gene transcript. An IRES (or other suitable sequence) is used to produce a protein that contains more than one polypeptide chain or to express two different proteins from or within the same cell. An exemplary IRES is the poliovirus internal ribosome entry sequence, which supports transgene expression in photoreceptors, RPE and ganglion cells. Preferably, the IRES is located 3′ to the transgene in the rAAV vector.

The selection of the promoter to be employed in the rAAV may be made from among a wide number of constitutive or inducible promoters that can express the CIP290 functional fragment transgene in the selected cell. In another embodiment, the promoter is cell-specific. The term “cell-specific” means that the particular promoter selected for the recombinant vector can direct expression of the selected transgene in a particular cell type.

In an embodiment in which the selected cell is a mammalian photoreceptor, the regulatory sequence comprises a promoter that expresses the product of the CEP290 fragment in mammalian photoreceptors. In one embodiment, the promoter is specific for expression of the transgene in photoreceptor cells. In another embodiment, the promoter is specific for expression in the rods and cones. In another embodiment, the promoter is specific for expression in the rods. In another embodiment, the promoter is specific for expression in the cones. In another embodiment, the promoter is specific for expression of the transgene in RPE cells. In another embodiment, wherein the selected cells is a mammalian cell is a kidney cell, brain cell or olfactory epithelium.

Suitable promoters for expression in photoreceptors may be selected from the rhodopsin promoter, the rhodopsin kinase promoter (51), the RPGTPase regulator (RPGR) promoter, the IRBP promoter, the GRK1 promoter. Alternatively, the promoter may be a constitutive promoter such as a chicken beta actin promoter, a chicken beta actin promoter with a cytomegalovirus enhancer, or modifications thereof, such as modification to shorten the length of the promoter.

In an embodiment in which the selected cell is a mammalian kidney cell, the regulatory sequence comprises a promoter that expresses the product of the CEP290 fragment in mammalian kidney cells. In an embodiment in which the selected cell is a mammalian brain cell or neuron of the CNS, the regulatory sequence comprises a promoter that expresses the product of the CEP290 fragment in mammalian CNS or brain cells. In an embodiment in which the selected cell is a mammalian bone cell, the regulatory sequence comprises a promoter that expresses the product of the CEP290 fragment in mammalian bone cells. In an embodiment in which the selected cell is a mammalian mucosal epithelial cell, such as olfactory epithelial cells, the regulatory sequence comprises a promoter that expresses the product of the CEP290 fragment in mammalian epithelial cells. Still other cellular targets suitable for expression of the CEP290 nucleic acid functional fragments may direct selection of the promoter.

The promoter may be derived from any species. In another embodiment, the promoter is the human G-protein-coupled receptor protein kinase 1 (GRK1) promoter (Genbank Accession number AY327580). In another embodiment, the promoter is a 292 nt fragment (positions 1793 to 2087) of the GRK1 promoter (See also, Beltran et al, Gene Therapy 2010 17:1162-74, which is hereby incorporated by reference herein). In another preferred embodiment, the promoter is the human interphotoreceptor retinoid-binding protein proximal (IRBP) promoter. In one embodiment, the promoter is a 235 nt fragment of the hIRBP promoter. In another embodiment, promoter is the native promoter for the gene to be expressed. In one embodiment, the promoter is the RPGR proximal promoter (Shu et al, IOVS, May 2012, which is incorporated by reference herein). Other promoters useful in the invention include, without limitation, the rod opsin promoter, the red-green opsin promoter, the blue opsin promoter, the cGMP-β-phosphodiesterase promoter, the mouse opsin promoter (Beltran et al 2010 cited above), the rhodopsin promoter (Mussolino et al, Gene Ther, July 2011, 18(7):637 to 45); the alpha-subunit of cone transducin (Morrissey et al, BMC Dev, Biol, January 2011, 11:3); beta phosphodiesterase (PDE) promoter; the retinitis pigmentosa (RP1) promoter (Nicord et al, J. Gene Med, December 2007, 9(12):1015-23); the NXNL2/NXNL1 promoter (Lambard et al, PLoS One, Octboer 2010, 5(10):e13025), the RPE65 promoter; the retinal degeneration slow/peripherin 2 (Rds/perph2) promoter (Cai et al, Exp Eye Res. 2010 August; 91(2):186-94); and the VMD2 promoter (Kachi et al, Human Gene Therapy, 2009 (20:31-39)). Each of these documents is incorporated by reference herein. In one embodiment, the promoter is of a small size, under 1000 bp, due to the size limitations of the AAV vector. In another embodiment, the promoter is under 400 bp.

Examples of constitutive promoters useful in the invention include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer), the SV40 promoter, the dihydrofolate reductase promoter, the chicken β-actin (CBA) promoter, the phosphoglycerol kinase (PGK) promoter, the EF1 promoter (Invitrogen), and the immediate early CMV enhancer coupled with the CBA promoter (Beltran et al, Gene Therapy 2010 cited above).

Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state, e.g., acute phase, a particular differentiation state of the cell, or in replicating cells only. Inducible promoters and inducible systems are available from a variety of commercial sources, including, without limitation, Invitrogen, Clontech and Ariad. Many other systems have been described and can be readily selected by one of skill in the art. Examples of inducible promoters regulated by exogenously supplied compounds, include, the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system; the ecdysone insect promoter, the tetracycline-repressible system, the tetracycline-inducible system, the RU486-inducible system and the rapamycin-inducible system. Other types of inducible promoters which may be useful in this context are those which are regulated by a specific physiological state, e.g., temperature, acute phase, a particular differentiation state of the cell, or in replicating cells only. Any type of inducible promoter which is tightly regulated and is specific for the particular target ocular cell type may be used.

Other regulatory sequences useful in the invention include enhancer sequences. Enhancer sequences useful in the invention include the IRBP enhancer (Nicord 2007, cited above), immediate early cytomegalovirus enhancer, one derived from an immunoglobulin gene or SV40 enhancer, the cis-acting element identified in the mouse proximal promoter, etc.

Selection of these and other common vector and regulatory elements are conventional and many such sequences are available. See, e.g., Sambrook et al, and references cited therein at, for example, pages 3.18 to 3.26 and 16.17 to 16.27 and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989). Of course, not all vectors and expression control sequences will function equally well to express all of the transgenes of this invention. However, one of skill in the art may make a selection among these, and other, expression control sequences without departing from the scope of this invention.

The Pharmaceutical Carrier and Pharmaceutical Compositions

The compositions of the present invention containing the recombinant viral vector, e.g., AAV, containing the desired transgene and cell-specific promoter for use in the selected target cells, e.g., photoreceptor cells for treatment of LCA, as detailed above is preferably assessed for contamination by conventional methods and then formulated into a pharmaceutical composition intended for a suitable route of administration. Still other compositions containing the desired CEP290 fragment, e.g., naked DNA or as protein, may be formulated similarly with a suitable carrier. Such formulation involves the use of a pharmaceutically and/or physiologically acceptable vehicle or carrier, particularly directed for administration to the target cell. In one embodiment, carriers suitable for administration to the photoreceptor cells of the eye include buffered saline, an isotonic sodium chloride solution, or other buffers, e.g., HEPES, to maintain pH at appropriate physiological levels, and, optionally, other medicinal agents, pharmaceutical agents, stabilizing agents, buffers, carriers, adjuvants, diluents, etc.

For injection, the carrier will typically be a liquid. Exemplary physiologically acceptable carriers include sterile, pyrogen-free water and sterile, pyrogen-free, phosphate buffered saline. A variety of such known carriers are provided in U.S. Pat. No. 7,629,322, incorporated herein by reference. In one embodiment, the carrier is an isotonic sodium chloride solution. In another embodiment, the carrier is balanced salt solution. In one embodiment, the carrier includes tween. If the virus is to be stored long-term, it may be frozen in the presence of glycerol or Tween20.

In other embodiments, e.g., compositions containing the CEP290 protein or amino acid, e.g., the single reading frame protein of SEQ ID NO: 6, the pharmaceutical compositions include a surfactant. Useful surfactants, such as Pluronic F68 ((Poloxamer 188), also known as Lutrol® F68) may be included with the vectored CEP290 as they prevent AAV from sticking to inert surfaces and thus ensure delivery of the desired dose.

As an example, one illustrative composition designed for the treatment of LCA comprises a recombinant adeno-associated vector carrying a nucleic acid sequence encoding a fragment of the CEP290 gene lacking at least one of its N-terminal and C-terminal inhibitory regions, under the control of regulatory sequences which express the product of said gene in a photoreceptor cell of a mammalian subject, and a pharmaceutically acceptable carrier. In one embodiment, the AAV is AAV8b. In another embodiment, the AAV is AAV 2/5. In another embodiment, the CEP290 nucleic acid fragment is any of the fragments described above or one or more of the fragments spliced together or otherwise administered together. One such combination is the minigene of SEQ ID NO: 5. The carrier is isotonic sodium chloride solution and includes a surfactant Pluronic F68.

In yet another exemplary embodiment, the composition comprises a recombinant AAV2/5 pseudotyped adeno-associated virus carrying a nucleic acid sequence encoding aa 1695 to 1965 of CEP290, the nucleic acid sequence under the control of a rhodopsin kinase promoter which directs expression of the product of said gene in said photoreceptor cells, wherein the composition is formulated with a carrier and additional components suitable for subretinal injection.

In yet another exemplary embodiment, the composition comprises simply unvectored naked minigene CEP290 or other fragments or spliced variant of CEP290 having the functions of natural or wildtype CEP290 formulated with a carrier.

Methods of Treatment/Prophylaxis

The compositions described above are useful in methods of treating a mammalian subject having a disease associated with a CEP290 mutation. These methods comprise administering to a subject in need thereof subject an effective concentration of a composition of any of those described herein.

In one illustrative embodiment, such a method is provided for preventing, arresting progression of or ameliorating vision loss associated with Lebers Congenital Amaurosis in a subject, said method comprising administering to a mammalian subject in need thereof an effective concentration of a composition comprising a recombinant adeno-associated virus (AAV) carrying a nucleic acid sequence encoding a fragment of the CEP290 gene lacking at least one of its N-terminal and C-terminal inhibitory regions, under the control of regulatory sequences which express the product of said gene in a photoreceptor cell of a mammalian subject, and a pharmaceutically acceptable carrier.

By “administering” as used in the methods means delivering the composition to the target selected cell based on the disease caused by CEP290 mutation or defect, and on the selected cell. For example, in one embodiment, the method involves delivering the composition by subretinal injection to the photoreceptor cells or other ocular cells. In another embodiment, intravitreal injection to ocular cells or injection via the palpebral vein to ocular cells may be employed. In another embodiment, the method involves delivering the composition via retrograde urethral injection to the kidney. In still another embodiment, the method involves delivering the composition by intra-ventricular or intra-cerebral injection to the brain. In yet a further embodiment, the method involves topically delivering the composition to nasal epithelium. Still other methods of administration may be selected by one of skill in the art given this disclosure.

Furthermore, in certain embodiments of the invention it is desirable to perform non-invasive retinal imaging and functional studies to identify areas of retained photoreceptors to be targeted for therapy. In these embodiments, clinical diagnostic tests are employed to determine the precise location(s) for one or more subretinal injection(s). These tests may include electroretinography (ERG), perimetry, topographical mapping of the layers of the retina and measurement of the thickness of its layers by means of confocal scanning laser ophthalmoscopy (cSLO) and optical coherence tomography (OCT), topographical mapping of cone density via adaptive optics (AO), functional eye exam, etc. In view of the imaging and functional studies, in some embodiments of the invention one or more injections are performed in the same eye in order to target different areas of retained photoreceptors.

For use in these methods, the volume and viral titer of each injection is determined individually, as further described below, and may be the same or different from other injections performed in the same, or contralateral, eye, where the disease is LCA. In another embodiment, a single, larger volume injection is made in order to treat the entire eye. Where the diseases are related to CEP290 expression or defect in another organ, the dosages, administrations and regimens may be determined by the attending physician given the teachings of this specification.

In one embodiment, the volume and concentration of the rAAV composition is selected so that only the certain regions of photoreceptors is impacted. In another embodiment, the volume and/or concentration of the rAAV composition is a greater amount, in order reach larger portions of the eye. Similarly dosages are adjusted for administration to other organs.

An effective concentration of a recombinant adeno-associated virus carrying a nucleic acid sequence encoding the CEP290 nucleic acid functional fragment or minigene under the control of the selected promoter sequence ranges between about 10⁸ and 10¹³ vector genomes per milliliter (vg/mL). The rAAV infectious units are measured as described in S. K. McLaughlin et al, 1988 J. Virol., 62:1963. In another embodiment, the concentration ranges between 10⁹ and 10¹³ vector genomes per milliliter (vg/mL). In another embodiment, the effective concentration is about 1.5×10¹¹ vg/mL. In one embodiment, the effective concentration is about 1.5×10¹⁰ vg/mL. In another embodiment, the effective concentration is about 2.8×10¹¹ vg/mL. In yet another embodiment, the effective concentration is about 1.5×10¹² vg/mL. In another embodiment, the effective concentration is about 1.5×10¹³ vg/mL. It is desirable that the lowest effective concentration of virus be utilized in order to reduce the risk of undesirable effects, such as toxicity, and other issues related to administration to the eye, e.g., retinal dysplasia and detachment. Still other dosages in these ranges or in other units may be selected by the attending physician, taking into account the physical state of the subject, preferably human, being treated, including the age of the subject; the composition being administered, e.g., viral vector, AAV, naked DNA or protein-containing; and the particular CEP-290-related disorder, e.g., LCA, Joubert syndrome, Senior Loken Syndrome, or Meckel-Grüber syndrome; the targeted cell or organ type, and the degree to which the disorder, if progressive, has developed.

The composition may be delivered in a volume of from about 50 μL to about 1 mL, including all numbers within the range, depending on the size of the area to be treated, the viral titer used, the route of administration, and the desired effect of the method. In one embodiment, the volume is about 50 μL. In another embodiment, the volume is about 70 μL. In another embodiment, the volume is about 100 μL. In another embodiment, the volume is about 125 μL. In another embodiment, the volume is about 150 μL. In another embodiment, the volume is about 175 μL. In yet another embodiment, the volume is about 200 μL. In another embodiment, the volume is about 250 μL. In another embodiment, the volume is about 300 μL. In another embodiment, the volume is about 450 μL. In another embodiment, the volume is about 500 μL. In another embodiment, the volume is about 600 μL. In another embodiment, the volume is about 750 μL. In another embodiment, the volume is about 850 μL. In another embodiment, the volume is about 1000 μL.

The invention provides various methods of preventing, treating, arresting progression of or ameliorating the CEP290-related diseases or disorders, exemplified by LCA, including ocular diseases and retinal changes associated therewith. Such treatment can treat or prevent the advanced of loss of photoreceptor structure or function and blindness associated with LCA caused by a defect in CEP290. The expression of the functional fragment of CEP290 in the targeted cell can result in improvement of the subject's vision or an arrest of vision loss.

In another embodiment, the invention provides a method to prevent, or arrest photoreceptor function loss, or increase photoreceptor function in the subject. Photoreceptor function may be assessed using the functional studies described herein, e.g., ERG or perimetry, which are conventional in the art. As used herein “photoreceptor function loss” means a decrease in photoreceptor function as compared to a normal, non-diseased eye or the same eye at an earlier time point. As used herein, “increase photoreceptor function” means to improve the function of the photoreceptors or increase the number or percentage of functional photoreceptors as compared to a diseased eye (having the same ocular disease), the same eye at an earlier time point, a non-treated portion of the same eye, or the contralateral eye of the same patient.

For each of the described methods, the treatment may be used to prevent the occurrence of further damage or to rescue tissues or organ, e.g., eyes in a subject with LCA, having mild or advanced disease. As used herein, the term “rescue” means to prevent progression of the disease, e.g., to total blindness with LCA, prevent spread of damage to uninjured photoreceptor cells or to improve damage in injured photoreceptor cells.

Thus, in one embodiment, the composition is administered before disease onset. In another embodiment, the composition is administered prior to the initiation of photoreceptor loss. In another embodiment, the composition is administered after initiation of photoreceptor loss. In yet another embodiment, the composition is administered when less than 90% of the photoreceptors are functioning or remaining, as compared to a non-diseased eye.

In another embodiment of the invention, the method includes performing functional and imaging studies to determine the efficacy of the treatment. These studies include ERG and in vivo retinal imaging, as described in U.S. Pat. No. 8,147,823; in co-pending U.S. patent application No. 61/670,355 or 61/762,775, incorporated by reference. In addition visual field studies, perimetry and microperimetry, mobility testing, visual acuity, color vision testing may be performed.

In yet another embodiment of the invention, any of the above described methods is performed in combination with another, or secondary, therapy. The therapy may be any now known, or as yet unknown, therapy which helps prevent, arrest or ameliorate CEP290 mutations or defects or any of the effects associated therewith. The secondary therapy can be administered before, concurrent with, or after administration of the rAAV described above.

EXAMPLES

The examples below present evidence that CEP290 directly binds to the ciliary membrane through a highly conserved region in its N-terminus and to microtubules through a domain located near its C terminus. CEP290 activity was found to be regulated by autoinhibitory domains located within its N and C-termini, both of which were found to play a critical role in regulating ciliogenesis. Furthermore, the inventors determined that the microtubule-binding domain is completely disrupted in the rd16 mouse LCA model (17), resulting in significant deficits in cilium formation leading to retinal degeneration.

These findings implicate CEP290 both as a key structural component of the ciliary Y-links and as a terminal regulator in the pathway leading to ciliogenesis. This data provides the first evidence of a mechanistic and pathological basis for CEP290-related LCA and related ciliopathies and supports novel therapeutic methods. Restoration of cellular function by the method of this invention can be assessed in an animal model of the appropriate disease caused by CEP290 defect or mutation, such as the restoration of visual function in a subject with a CEP290 defect causing LCA in the rd16 mouse LCA model or canine model of LCA. The use of the exemplary vector can demonstrate that the defect in the mutant dog or other animal model could be corrected by gene delivery. This data allow one of skill in the art to readily anticipate that this method may be similarly used in treatment of XLRP or other types of retinal disease in other subjects, including humans.

Example 1—Materials and Methods Plasmid Construction

CEP290 truncations were generated by PCR amplification using Gateway® cloning (Invitrogen) compatible primers identified below in Table 1 from a human codon-optimized CEP290 plasmid synthesized by DNA 2.0. Amplified products were directly cloned into pDONR221 (Invitrogen) by Gateway® cloning to generate entry clones. For cell transfection and in vitro transcription and translation assays, entry clones were shuttled into the plasmid pcDNA-DEST53 (Invitrogen) by Gateway® LR clonase reactions to create N-terminally tagged GFP fusions.

TABLE 1 primers used to generate truncation mutants SEQ Truncation ID NO. Primer Sequence aa  7 Forward GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGAAGGAGATAGAA 1-2479 CCATGCCCCCAAACATCAATTGG  8 Reverse GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAATAGATCGGGA AGTTAACAGG aa  9 Forward GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGAAGGAGATAGAA 580to2479 CCATGACGGAGAACATAAGCCAAGG 10 Reverse GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAATAGATCGGGA AGTTAACAGG aa1 to 580 11 Forward GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGAAGGAGATAGAA CCATGCCCCCAAACATCAATTGG 12 Reverse GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAAAGATTCAGAT CCTCGGTAG aa 1-362 13 Forward GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGAAGGAGATAGAA CCATGCCCCCAAACATCAATTGG 14 Reverse GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAATCCCTTTCTT GGATTCCCTGC aa 15 Forward GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGAAGGAGATAGAA 380-580 CCATGAAAAACACTTGCATCATTGAGGAC 16 Reverse GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAAAGATTCAGAT CCTCGGTAG aa 1-1695 17 Forward GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGAAGGAGATAGAA CCATGCCCCCAAACATCAATTGG 18 Reverse GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAATCCAGCAGAT ACTTCAAATCC aa 19 Forward GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGAAGGAGATAGAA 580-1695 CCATGACGGAGAACATAAGCCAAGG 20 Reverse GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAATCCAGCAGAT ACTTCAAATCC aa 1-1966 21 Forward GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGAAGGAGATAGAA CCATGCCCCCAAACATCAATTGG 22 Reverse GGGGACCACTTTGTACAAGAAAGCTGGGTCCTATGTTTTCAGCT TTCTCTGCAG aa 23 Forward GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGAAGGAGATAGAA 580-2479 CCATGACGGAGAACATAAGCCAAGG 24 Reverse GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAATAGATCGGGA AGTTAACAGG aa 25 Forward GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGAAGGAGATAGAA 580-1966 CCATGACGGAGAACATAAGCCAAGG 26 Reverse GGGGACCACTTTGTACAAGAAAGCTGGGTCCTATGTTTTCAGCT TTCTCTGCAG aa 27 Forward GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGAAGGAGATAGAA 1695-1966 CCATGCAGTCCCAGAAGGAGTCAC 28 Reverse GGGGACCACTTTGTACAAGAAAGCTGGGTCCTATGTTTTCAGCT TTCTCTGCAG aa 29 Forward GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGAAGGAGATAGAA 1966-2479 CCATGACAGGCATGACCGTGGAC 30 Reverse GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAATAGATCGGGA AGTTAACAGG aa 31 Forward GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGAAGGAGATAGAA 1695-1903 CCATGCAGTCCCAGAAGGAGTCAC 32 Reverse GGGGACCACTTTGTACAAGAAAGCTGGGTCCTATTTCTCTTTCA TGGGCTTAAGGTC aa 33 Forward GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGAAGGAGATAGAA 1903-2479 CCATGACAGGCATGACCGTGGACC 34 Reverse GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAATAGATCGGGA AGTTAACAGG

For bacterial expression, entry clones were shuttled into pDest-527 (a gift of Dominic Esposito (Addgene plasmid #11518)) to create N-terminally-tagged 6×His fusions. For lentivirus production, entry clones were shuttled into pLXnGFP, a modified version of pLX302 (a gift of David Root (Addgene plasmid #25896)), to create N-terminally-tagged GFP fusion lentivirus production plasmids. pLXnGFP was created by the replacement of the gateway cassette of pLX302 with an eGFP cassette containing an EcoRV site just downstream of the eGFP ORF by BsrGI digestion and ligation. The Gateway A cassette was then inserted into the EcoRV site using the Gateway Conversion Kit (Invitrogen). Restriction digest and DNA sequencing were used to confirm the integrity of each expression construct.

shRNA constructs were created as follows. Three regions of the human CEP290 coding sequence without homology to other transcripts were selected as shRNA targets. Oligonucleotides encoding the target sequence in the context of a DNA hairpin were synthesized, annealed, and ligated into the pSIREN-RetroQ retrovirus vector (Clontech). Efficiency of knockdown was assayed. The mammalian expression vector encoding GFP-fused miniCEP290 was created by the amplification of the miniCEP290 gene by PCR using primers compatible with Gateway® cloning (Invitrogen). Amplified products were cloned into pDONR221 (Invitrogen) by Gateway® cloning to generate entry clones and subsequently shuttled into the plasmid pcDNA-DEST53 (Invitrogen) by Gateway® LR clonase reactions to create N-terminally-tagged GFP fusions.

MiniCEP290 Synthesis

The miniCEP290 construct was codon optimized, synthesized, and sequenced by DNA2.0.

Cell Culture and Treatments; Lentivirus Production; Retrovirus Production

Wild type and rd16 mouse primary dermal fibroblasts and human 293T cells were grown in DMEM supplemented with 10% FBS. hTERT RPE to 1 cells were grown in DMEM:F12 supplemented with 10% FBS and 0.075% sodium bicarbonate. All cells were grown at 37° C. in a humidified 5% CO₂ atmosphere. All transfections were performed with FuGENE 6 reagent (Promega) according to the manufacturer's protocol. Cells were induced to form primary cilia by serum starvation with OptitoMEM I (Invitrogen) for 48 to 72 hours.

Lentiviral vectors were produced by transfection of 80% confluent 293T cells grown in T25 culture flasks with 1 μg of lentivirus construct, 750 ng of PsPAX2 packaging plasmid, and 250 ng of pMD2.G envelope plasmid using FuGENE6. Media was replaced after 24 hours, and lentiviral supernatants were harvested at 48 and 72 hours after transfection, combined, filtered through a 0.45 μm filter, and snap-frozen at to 80° C. For lentivirus transduction, hTERT RPE-1 cells were plated in 6 well plates in media containing 8 μg/mL polybrene. 0.5 mL of filtered media containing the appropriate lentivirus particles was added to each well. 24 hours after transduction cells were switched to media containing 10 μg/mL puromycin and maintained in selective media from that point on. All experiments carried out on lentivirus transduced cells were performed 10 days post transduction.

Retroviral vectors were produced by transfection of 80% confluent 293T cells in 100 mm culture dishes with 10 μg of retrovirus construct and 10 μg of pCL10A1 packaging plasmid. Media was replaced after 24 hours, and retroviral supernatants were harvested at 48 and 72 hours, combined, filtered through a 0.45 μm filter, and snap-frozen at −80° C. For retrovirus transduction, hTERT RPE-1 cells were plated in media containing 8 μg/mL polybrene. Filtered media containing the appropriate retroviral particles was added and 24 hours after transduction cells were switched to selective media containing 10 μg/mL puromycin.

Primary Dermal Fibroblast Isolation

Primary dermal fibroblasts were isolated by washing sections of neonatal mouse skin in 70% ethanol followed by 5 washes in PBS. The skin was minced and applied to the bottom of a culture vessel, covered with DMEM supplemented with 10% FBS, penicillin, and streptomycin, and incubated at 37° C. in a humidified 5% CO2 atmosphere. One week after harvesting the skin was removed and discarded and fibroblasts were passaged into a larger vessel.

Antibodies, Immunofluorescence, and Immunoblotting

Antibodies used in this study were rabbit anti-human CEP290 (Abcam, ab105383), rabbit anti-mouse Cep290 (Abcam, ab128231), mouse anti-α tubulin (Abcam, AB7291), rabbit anti-pericentrin (Abcam, AB4448), mouse anti-GFP (Roche 11 814 460 001), mouse anti-Bovine serum albumin (Thermo, MA5 to 15238), rabbit anti-6×His (Abcam, AB1187), rabbit anti-GAPDH (Sigma, SAB2103104), mouse anti-Na/K ATPase α-1 (Novus, NB300 to 146), mouse anti-Lamin A/C (Sigma, SAB4200236), mouse anti-Acetylated α-tubulin (Sigma, T7451), rabbit anti-LAMP2 (Novus, NBP1 to 71692), rabbit anti-Annexin A2 (Cell Signaling, 8235), rabbit anti-ARL13B (Proteintech, 17711-1-AP), HRP-conjugated goat anti-mouse (GE, NA931V), HRP-conjugated goat anti-rabbit (GE, NA934V), Cy5 conjugated goat anti-rabbit (KPL, 072-02-15-06), and AlexaFluor 594-conjugated goat anti-mouse (Invitrogen, A1100S).

For immunofluorescence, cells were grown in chamber slides and fixed with 3% PFA in PBS for 20 minutes at 37° C. Cells were permeabilized with 1% Triton X-100 in PBS for 5 minutes, and blocked in 2% BSA in PBS for 30 minutes prior to incubation with primary antibody. Secondary antibodies used were donkey anti-mouse or anti-rabbit, conjugated to Cy5 or AlexaFluor 594. Slides were mounted in mounting medium containing DAPI. Confocal imaging was performed with an LSM510 META NLO laser scanning confocal on a Zeiss Axiovert 200M inverted microscope using a Plan-Apo 63×/1.4 oil objective and the LSM510 4.2 software. Laser lines used were 488 nm (for green labeling, from argon laser), 543 nm (for red labeling, HeNe laser), 633 nm (for Cy5 channel, HeNe laser), and 740 nm (for DAPI channel, from a Coherent Chameleon tunable two photon laser). For normal fluorescence microscopy, slides were imaged using an Axio Imager.M2 microscope using an either EC Plan-Neofluar 40×/0.75 M27 or an EC Plan-Neofluar 63×/1.25 Oil M27 objective and captured using an AxioCamMR3 camera and the AxioVs40 software, version 4.8.2.0. Primary cilium length was measured using the same AxioVs40 software.

For immunoblotting, samples were subjected to SDS-PAGE and transferred to nitrocellulose membrane using standard techniques. Membranes were blocked in 5% nonfat milk for 1 hour at room temperature and subsequently incubated in primary antibody overnight at 4° C. Membranes were washed three times with PBST (0.1% TweeN-20 in PBS) and incubated with HRP-conjugated secondary antibody for 1 hour at room temperature. Membranes were washed three times with PBST, developed using ECL2 reagent (Pierce), and scanned on a Typhoon 9400 instrument (GE). Immunoblots were quantified by densitometry using ImageJ 1.44p.

Membrane Flotation, Membrane Protein Fractionation, and Vesicle Immunoprecipitation

Cultured cells were washed three times with ice cold PBS. Cells were then resuspended in a 250 mM Sucrose solution containing 4 mM Immidazole and a protease inhibitor cocktail and passaged through a 25 G needle 20 times to rupture the plasma membrane. The resulting lysate was centrifuged at 1,000×g for 10 minutes at 4° C. to pellet nuclei and unlysed cells and the resulting post-nuclear supernatant was centrifuged at 100,000×g for 60 minutes at 4° C. to pellet the membrane-enriched fraction. Membrane flotation was performed by resuspending the membrane-enriched fraction in 250 μL of 80% sucrose in PBS. This solution was added to the bottom of a 2 mL centrifuge tube, overlaid with 1.5 mL of 50% sucrose in PBS, and in turn overlaid with 250 μL of 5% sucrose in PBS. Sucrose gradients were centrifuged at 100,000×g for 16 hours at 4° C. to induce the membrane and membrane associated proteins to float to the top of the gradient. Equal fractions were subsequently taken from the top to the bottom of the gradient and analyzed by western blotting as indicated. Peripheral membrane proteins were extracted from membrane preparations by resuspending and incubating the membrane-enriched fraction of cultured cells in a high pH buffer (100 nM Na2CO3, pH 11.3) for 30 minutes at 4° C. The remaining membranes were pelleted at 100,000×g for 60 minutes at 4° C. and the resulting supernatant was saved as the peripheral membrane fraction. Integral membrane proteins were subsequently extracted by resuspending and incubating the resulting membrane pellet in 4% TritoN-X100 in PBS for 30 minutes at 4° C. Both fractions were analyzed by immunoblotting for the CEP290 truncations being tested and for the indicated fractionation controls. Vesicle immunoprecipitation was performed on post-nuclear supernatants of hTERT-RPE1 cells expressing various CEP290 truncations.

A sample of the total post nuclear extract was saved as the input fraction. Protein G Dynabeads (Invitrogen) were washed and incubated with 2 μg of anti-ARL13B antibody for 20 minutes at room temperature, magnetically collected, and washed three times with PBS. 350 μL of post-nuclear supernatant was added to the antibody-dynabead complex and incubated with gentle agitation for 20 minutes at room temperature. The beads and immunoprecipitated complexes were magnetically collected and washed three times with PBS. A sample of the unbound fraction was saved and the immunoprecipitated material was eluted by resuspension in 4×SDS PAGE sample buffer. Samples were analyzed by immunoblotting.

Subcellular Fractionation

Subcellular fractionation was performed using the QProteome Cell Compartment kit (Qiagen) according to the supplied protocol. All fractions were analyzed by immunoblotting for the CEP290 truncations being tested and for the indicated fractionation controls and quantified by densitometric analysis using ImageJ 1.44p.

Recombinant Protein Expression and Purification

6×His tagged CEP290 truncation M and N were expressed from pDest-527 in E. coli BL21(DE3)pLysS (Invitrogen) and purified using the Ni-NTA Fast Start Kit (Qiagen) according to the manufacturer's protocol. Purified protein products were subjected to SDS-PAGE and stained with Coomassie Brilliant Blue to assess purity and determine protein concentration.

Liposome Flotation Assay

100 nm liposomes (total lipid concentration of 5 mg/mL in PBS) with a lipid composition of a 60:40 molar ratio of phosphatidylserine to cholesterol were purchased from Encapsula NanoSciences and used within 2 weeks of their formulation. 20 μL (1.5 μg) of recombinant CEP290 truncation N, or an equivalent amount of BSA, was incubated with 230 μL of liposomes, or an equal volume of PBS alone, at 37° C. for 30 minutes. Each reaction was then mixed 1:1 with a solution of 80% sucrose in PBS and added to the bottom of a 2 mL ultracentrifuge tube. Reactions were overlaid with 1.3 mL of 30% sucrose in PBS, which was in turn overlaid with 200 μL of PBS. Liposomes and liposome associated protein were induced to float to the top of the gradient by centrifugation at 100,000×g for 90 minutes at 30° C. Five 400 μL fractions were taken, starting at the top of sucrose gradient, and equal amounts of each were analyzed by SDS-PAGE and western blotting, probing for either the 6×His tag or BSA.

In Vitro Transcription and Translation Reactions

Plasmid DNA was transcribed and translated using the TNT T7 reticulocyte lysate system (Promega) according to the manufacturer's protocol.

Microtubule Polymerization

Pure bovine tubulin (Cytoskeleton) was diluted 1:1 in BRB80 buffer (80 mM Pipes, 1 mM MgCl₂, 1 mM EGTA, pH 6.8) and cleared of insoluble material by centrifugation at 20,000×g for 10 minutes. The soluble fraction was supplemented with 1 mM GTP and incubated at 37° C. for 15 minutes to polymerize microtubules. The polymerized microtubules were then treated with 10 μM taxol and incubated at room temperature for a further 15 minutes to stabilize the microtubules. Microtubules were then pelleted at 48,000×g for 30 minutes at 30° C. and resuspended in BRB80 supplemented with 1 mM GTP and 10 μM taxol. Microtubules were used within one week of preparation.

Microtubule Binding Assays

Crude TNT T7 reaction products were diluted 1:1 in BRB80 supplemented with 1 mM GTP and 10 μM taxol. The diluted products were then incubated at 30° C. for 30 minutes in either the presence or absence of pure, pre-polymerized microtubules. Reactions were then centrifuged through a 40% sucrose cushion at 48,000×g for 30 minutes, the supernatant was collected, and the pellet washed once with warm BRB80 and resuspended in 1×SDS PAGE sample buffer. Both fractions were subjected to SDS-PAGE and transferred to nitrocellulose. The presence of tubulin in the pellets was confirmed by Ponceau staining, and GFP-tagged CEP290 constructs were detected by immunoblotting.

For microtubule binding assays performed on mouse brain homogenate, 0.5 g of mouse brain was mechanically homogenized in 0.5 mL of 1% NP40 in BRB80 buffer containing a protease inhibitor cocktail. The homogenate was cleared of insoluble material by centrifugation at 48,000×g for 30 minutes at 4° C., and the resulting supernatant was either used immediately or snap-frozen at to 80° C. for later use. Homogenates were incubated either at 37° C. for 30 minutes with 1 mM GTP and 10 μM taxol to promote microtubule polymerization, or at 4° C. for 30 minutes to inhibit microtubule polymerization. The resulting reactions were then layered over a 40% sucrose cushion, centrifuged at 48,000×g for 30 minutes to pellet the microtubules and microtubule-associated proteins, and processed as above.

For the direct microtubule binding assay, 1 μM of recombinantly expressed and purified 6×His tagged CEP290 truncation M was incubated at 30° C. for 30 minutes with increasing amounts of pure, prepolymerized microtubules. Reactions were centrifuged and subjected to SDS-PAGE as above, and tubulin and CEP290 truncation M were detected by Coomassie blue staining.

Bioinformatic Analysis

The helical wheel projection in FIG. 2E was adapted from the Helical Wheel Projection applet available at rzlab.ucr.edu/scripts/wheel/wheel.cgi. The multiple sequence alignment was adapted from GeneDoc 2.7.000.

Statistical Analysis

The statistical significance of the difference between two means was determined using a two-tailed Student's t-test. The statistical significance of the difference between three or more means was determined using a two-way ANOVA and Tukey's HSD test. Statistical analysis was performed using GraphPad Prism Software 5.0b. p-values<0.05 were considered significant.

Example 2—CEP290 Associates with ARL13B Positive Cellular Vessicles Via its N-Terminus

Mutations in the CEP290 gene have been implicated in a variety of human diseases, but their effects on protein function have not yet been characterized. Mutations clustering in particular regions of the gene might be indicative of important functional domains, but no mutational hotspots or functional domains have been identified to date (FIG. 1A). To understand the role CEP290 plays in cilium function, ciliogenesis, and human disease, a structure-function analysis was performed using a panel of truncation constructs spanning the full length of CEP290 in order to identify and define domains of novel functionality.

The first two CEP290 truncations displayed distinct localization patterns. When overexpressed as GFP fusion proteins, the N-terminal fragment of CEP290 (spanning aa 1 to 580) showed an exclusively vesicular localization pattern while the C-terminal fragment of CEP290 (from aa 580 to the end of the protein, aa 2479) (SEQ ID NO: 60) showed a striking fibrillar localization pattern (FIG. 1B). Both of these patterns were occasionally observed in cells overexpressing the full length CEP290 construct, but not with the same frequency as in cells expressing the truncations (data not shown).

Two additional truncations of the N-terminal region of the protein were generated to better define the domain responsible for the vesicular localization. The truncation spanning CEP290 aa 1 to 362 showed a vesicular pattern of localization similar to that observed for the complete N-terminal fragment, while the truncation spanning aa 380 to 580 (SEQ ID NO: 61) was present only diffusely throughout the cytoplasm (FIG. 1B). Further truncation of the protein was not effective in resolving its membrane association property beyond aa 1 to 362, implying either that this is the minimum region needed for CEP290 membrane association or that further truncation significantly interferes with protein function.

The CEP290-positive vesicles observed by microscopy were found to display robust co-staining with ARL13B (FIG. 1B), a membrane protein important in the trafficking of vesicles to the primary cilium. To biochemically assay this colocalization, detergent free post-nuclear supernatants of cells expressing our CEP290 truncations were prepared and ARL13B positive vesicles were magnetically immunoprecipitated and analyzed by western blotting. Both CEP290 aa 1 to 580 and CEP290 aa 1 to 362 were significantly enriched in the ARL13B immunoprecipitate, while neither CEP290 aa 380 to 580 nor GFP alone were found in significant quantities (FIG. 1D).

Thus, CEP290 aa 1 to 362 were found to be necessary and sufficient to mediate CEP290 localization to ARL13B positive cellular vesicles relevant to primary cilium biology. In only very few cases did the CEP290-positive vesicles exhibit any costaining with LAMP2, a marker of the lysosomal compartment (FIG. 2A), indicating that CEP290 vesicular localization was not an artifact of protein overexpression.

To confirm that the vesicular structures observed by microscopy were truly membranous organelles and not aggregates of overexpressed protein we performed a series of membrane co-flotation assays on cells expressing CEP290 aa 1 to 580. More than ⅓ of total CEP290 aa 1 to 580 was found to co-float in the membrane-associated fractions (FIG. 1C), indicating that a substantial portion of the protein was, in fact, associated with cellular membranes. ARL13B was found to co-float in the same fractions as CEP290 aa 1 to 580, corroborating the colocalization between the two that we had observed by microscopy and co-IP (FIG. 1C). The dual distribution of CEP290 aa 1 to 580 within both the membrane-associated and soluble fractions along with the absence of a signal peptide from CEP290's amino acid sequence was suggestive of peripheral, rather than integral, membrane association. To confirm this, membrane fractions of cells transfected with our CEP290 truncations were prepared and peripheral membrane proteins were eluted from the membrane with a high pH buffer. The remaining integral membrane proteins were subsequently solubilized with detergent. For both CEP290 aa 1 to 580 and aa 1 to 362 the majority of each truncation was found in the peripheral membrane protein fraction (FIG. 1E). This same pattern was observed for the peripheral membrane protein Annexin A2 (36), while the majority of the Na/K ATPase, an integral membrane protein, was found in the integral membrane protein fraction (FIG. 1E). CEP290 aa 380 to 580, on the other hand, was not found in significant amounts in either fraction. Taken together, these data indicate that CEP290 aa 1 to 362 are necessary and sufficient for robust peripheral membrane association.

Example 3—CEP290's Capacity for Membrane Association is Increased by Truncation of its C-Terminus

To further investigate CEP290's membrane association, a series of subcellular fractionation experiments on cells expressing each of our CEP290 truncations was performed. The CEP290 truncation spanning aa 580 to 2479 that produced a fibrillar localization pattern by microscopy was found almost exclusively in the cytoskeletal fraction, while truncations lacking this region were completely absent from the cytoskeletal fraction (FIG. 2B). On the other hand, truncations that included CEP290 aa 1 to 362 were again found to be significantly present in the membrane fraction when compared to either GFP alone or our fractionation controls (FIG. 2B). For both CEP290 aa 1 to 580 and aa 1 to 362, roughly 30% of each truncation was found to be associated with cellular membranes (FIG. 2C), demonstrating again that CEP290 aa 1 to 362 are necessary and sufficient for membrane association. Interestingly, a small amount of full length CEP290 was also found in the membrane fraction (FIG. 2B to C). This distribution agrees with what was observed by fluorescence microscopy—specifically, that full length CEP290 occasionally displayed vesicular localization, but to a lesser extent than CEP290 truncations lacking the C-terminus of the protein but containing aa 1 to 362.

Example 4—Cep290 Directly Binds Membranes In Vitro and Contains a Highly Conserved Membrane Binding Amphipathic α-Helix Motif

To determine whether CEP290's membrane association was mediated by a direct or indirect membrane interaction a series of liposome co-flotation assays were performed on purified recombinant CEP290 aa 1 to 580. CEP290 aa 1 to 580 associated with liposomes robustly, with a majority of the protein found in the liposome-associated fraction (FIG. 2D). Flotation of the truncation occurred only in the presence of liposomes, and liposome co-flotation was not observed for a control protein, BSA (FIG. 2D). The ability of this region of CEP290 to directly bind liposomes suggests that the observed association between the N-terminus of CEP290 and cellular membranes is mediated by a direct interaction.

Projecting CEP290 aa 1 to 362 onto an α-helical wheel indicated that a segment from aa 257 to 292 was predicted to form a canonical amphipathic α-helix (FIG. 2E). Such helices have been shown to be critical in mediating robust interactions between peripheral membrane proteins and various cellular membranes. Comparing this stretch of the protein across a variety of species, we found the amphipathic helix motif to be very highly conserved. An examination of a sequence alignment of aa257 to 292 from a variety of species shows that where there was divergence in the amino acid sequence from the sources of CEP290 aa 257 to 292 for the following species: Gallus SEQ ID NO: 35, Meleagris SEQ ID NO: 36, Rattus SEQ ID NO: 37, Mus SEQ ID NO: 38, Pongo SEQ ID NO: 39, Macaca SEQ ID NO: 40, Homo sapiens SEQ ID NO: 41, Felis SEQ ID NO: 42, Ailuropoda SEQ ID NO: 43 and Danio SEQ ID NO: 45, there was usually conservation of polarity and charge between the divergent residues. Taken together, these data support that the highly conserved amphipathic helix located within the membrane binding region of CEP290 mediates CEP290's novel membrane binding function.

Example 5—CEP290 aa 1695 to 1966 Mediate Colocalization with Microtubules

CEP290 aa 580 to 2479 appeared both by microscopy and subcellular fractionation to be associated with the cytoskeleton (FIG. 1B, 2A). To further investigate this phenomenon, we constructed a library of additional CEP290 truncations to thoroughly interrogate CEP290's cytoskeletal association (FIG. 3B). Overexpression of these truncations as GFP fusions revealed that those truncations containing CEP290 aa 1695 to 1966, referred to as region M, displayed a fibrillar localization pattern similar to that which was observed for CEP290 aa 580 to 2479 (FIG. 3A, B). The fibrils formed by these CEP290 truncations were noted to co-localize with the microtubule network. Truncations lacking CEP290 region M never display any fibrillar localization (FIG. 3A, B). This along with the fact that CEP290 region M alone showed robust colocalization with the tubulin network implicated CEP290 region M as necessary and sufficient for microtubule co-localization.

The degree to which different CEP290 truncations displayed a fibrillar localization pattern was found to be dependent upon which regions of the protein were included in the truncation. The full length CEP290 construct was noted to produce a fibrillar localization pattern in only about 10% of transfected cells (FIG. 3C). Truncations lacking either the N- or C-terminus of CEP290, on the other hand, were found to display fibrillar localization in roughly 20% and 60% of transfected cells, respectively. The truncation lacking both termini was found to display a fibrillar localization pattern in nearly 80% of transfected cells. These data indicated that the N- and C-termini of the protein have an inhibitory or regulatory effect on CEP290's microtubule binding ability. All of our truncations displayed co-localization with pericentrin, a marker of the centriole (the normal site of CEP290 localization) (FIG. 3A, insets). Homotypic interactions between endogenous CEP290, present at the centrioles, and our truncations (through CEP290's homo/heterodimerization domains, found within either terminus of the protein (29)) might explain the observed centriolar localization of a number of our truncations. However, those truncations lacking both homo/heterodimerization domains are apparently still capable of localizing to the centriole, implying that multiple regions throughout CEP290 are capable of affecting centriolar localization.

Example 6—CEP290 Microtubule Association Results in Microtubule Acetylation and Bundling

In cells transfected with CEP290 constructs containing region M there was a dramatic increase in the intensity of acetylated α-tubulin staining, with bundles of acetylated microtubules looping throughout the cells (FIG. 4A). These increases were not seen for truncations lacking region M. The degree to which different CEP290 truncations increased the acetylation and bundling of microtubules was dependent upon which regions of the protein were included in the truncation. Full length CEP290 and CEP290 truncations lacking either the N- or C-terminus increased microtubule acetylation and bundling in nearly 40% of transfected cells while CEP290 truncations lacking both the N- and C termini of the protein increased microtubule acetylation and bundling in nearly 75% of cells (FIG. 4B). Less than 15% of cells transfected with truncations lacking region M were noted to have any change in microtubule acetylation or bundling.

Example 7—CEP290 Directly Binds Microtubules In Vitro

To test whether microtubule co-localization was indicative of an interaction between CEP290 and microtubules, we performed a series of in vitro microtubule co-sedimentation assays using our CEP290 truncation constructs. Truncations containing CEP290 region M were found to significantly associate with microtubules in vitro, while those truncations lacking this region displayed no significant microtubule association (FIG. 4C, D). Thus, region M was found to be necessary and sufficient to mediate robust CEP290 microtubule association. Again, the degree to which different CEP290 truncations associated with microtubules was found to be dependent on the inclusion of the N- and C-termini. Less than 50% of the full length CEP290 constructs and CEP290 constructs lacking either terminus were found to associate with microtubules, while nearly 100% of CEP290 constructs lacking both termini associated with microtubules (FIG. 4D). To test whether CEP290 region M's microtubules association was mediated by direct microtubule binding we recombinantly expressed and purified CEP290 region M and subjected this protein to a series of microtubule co-sedimentation assays using increasing concentrations of microtubules (FIG. 4E, F). Region M was found to directly and robustly bind to microtubules in a concentration dependent manner (FIG. 4F). The calculated KD of this interaction was found to be approximately 100 nM, an affinity comparable to those of other microtubule binding proteins (38, 39).

Example 8—the N- and C-Termini of CEP290 Cooperate to Inhibit Protein Function and Regulate Ciliogenesis

The observation that the N- and C-termini of CEP290 appeared to act in inhibiting the membrane and microtubule binding activity of the protein (FIG. 2B, 3C, 4B, 4D) suggested that these regions might be regulatory domains mediating the autoinhibition of the protein. To test this hypothesis, we transduced hTERT-RPE1 cells with lentiviral vectors encoding either the N- or C-terminal regulatory regions of the protein and observed the cells for deficits in primary cilium formation. To our surprise, we found that overexpression of either of these regulatory regions resulted not in deficiencies in primary cilium formation, but instead in significant increases in the percent of cells forming primary cilia, with more than twice as many cells forming primary cilia than those cells treated with a control vector (FIG. 5A, B). The length of cilia formed by cells overexpressing either regulatory region was also significantly increased by more than 25% compared to cells treated with the control vector (FIG. 5C, D). These increases were only observed in cells maintained in media supplemented with serum, a condition in which CEP290 is normally inhibited (24), implying that these regulatory domains act through the same pathway that mediates normal CEP290 inhibition. Dysregulation of CEP290 by overexpression of either regulatory region was sufficient to initiate aberrant primary cilium formation, suggesting that there is no further downstream regulation of ciliogenesis beyond CEP290. Interestingly, in both the serum starved and serum fed state it was noted that occasional cells transduced with the N-terminus of CEP290 appeared to produce multiple ciliary axonemes at the same centrosome (FIG. 5A, 5E to 5G). These axonemes always emanated from a single focus of pericentrin (FIG. 5F) and were often found at 90° to each other. In some, but not all cases, one or more of these axonemes co-stained with ARL13B (FIG. 5E), a protein associated with the ciliary membrane (32), indicating that at least some of these multitoaxoneme structures were fully formed cilia.

Example 9—the RD16 Mouse CEP290 Gene Encodes a Version of the Protein Deficient in Microtubule Binding

The rd16 mouse is a retinal disease model of CEP290 deficiency characterized by a rapid and near complete degeneration of photoreceptors. The rd16 mouse Cep290 gene encodes a Cep290 protein containing an in-frame deletion of 298 amino acids (17) that overlaps the region of human CEP290 we identified as being critical for microtubule binding (FIG. 6A). We generated two truncation mutants of human CEP290 containing either the region of the microtubule binding domain deleted in the rd16 mouse, or the region of the microtubule binding domain spared by the mouse deletion (FIG. 6A) to test whether microtubule binding might be impaired by the rd16 deletion. To our surprise, when overexpressed in hTERT-RPE1 cells, both truncations displayed a diffuse localization pattern indicative of a primarily cytosolic localization (FIG. 6B).

In neither case was any significant co-localization with the microtubule network observed. We confirmed that neither of these constructs was capable of associating with microtubules by subjecting them to microtubule co-sedimentation assays (FIG. 6C). Neither construct was found to significantly co-sediment with microtubules compared to the no-microtubule control (FIG. 6D), indicating both that the rd16 deletion perturbs microtubule binding and that microtubule binding is conferred by a larger portion of the CEP290 gene than was included in either of our truncations.

To confirm that rd16 Cep290 was in fact deficient in microtubule binding we subjected brain lysates from wild type (WT) and rd16 mice to microtubule co-sedimentation assays (FIG. 6E). WT Cep290 showed very significant microtubule association, with roughly 60% of the protein associating with microtubules (comparable to full length human CEP290 (FIG. 4D)), while rd16 Cep290 was found to be completely deficient in microtubule binding (FIG. 6F).

Example 10—the Rd16 Mouse is Deficient in Cilium Formation and Structure

While the retinal phenotype of the rd16 mouse has been well documented (40), no cellular phenotype regarding primary cilium formation or structure has yet been reported. To investigate the effect that ablation of Cep290 microtubule binding might have on primary cilium formation, we assayed primary dermal fibroblasts from rd16 and WT mice for deficiencies in cilium formation and structure. In both serum starved and serum fed conditions, rd16 fibroblasts were found to be significantly deficient in primary cilium formation, with roughly 50% fewer cells forming cilia than WT controls (FIG. 7A to C). The cilia produced by rd16 fibroblasts were also found to be more than 25% shorter than those produced by WT fibroblasts (FIG. 7D, E), further suggesting that the microtubule binding functionality of CEP290 is critically important in the maintenance and formation of the primary cilium and, when disrupted, capable of causing severe retinal disease.

Discussion of Examples 1 to 10

CEP290 acts as a bridge between the ciliary membrane and the microtubule axoneme. To date, how CEP290 functions as a component of the ciliary Y-links has been unclear. The inventors have shown that CEP290 is capable of directly binding to both ciliary membranes and microtubules, anchoring the two to each other and likely playing a key structural role in the maintenance of the cilium. Furthermore, particular domains of CEP290 responsible for mediating these specific functions have been identified. The N-terminus of the protein, containing a highly conserved amphipathic helix, mediates CEP290's membrane binding activity. Multiple experiments determined that a region near the C-terminus of CEP290, encompassing much of the protein's myosin-tail homology domain, was necessary and sufficient to mediate microtubule binding.

The location of these two functional domains at opposite ends of CEP290 immediately supports an important structural role for the protein, anchoring the ciliary membrane to the axoneme at a fixed distance, which is likely critical to the process and regulation of IFT. In addition to binding microtubules, CEP290 is capable of mediating their acetylation and bundling, two hallmarks of the microtubules that make up the ciliary axoneme, suggesting that CEP290 likely plays an important role in the stabilization, bundling, and organization of microtubules during ciliogenesis.

This data and analysis of CEP290's membrane and microtubule binding activities provides evidence that the full length protein exhibited attenuated activity when compared to truncation mutants lacking the N- or C-terminus. Confirming a role for these domains in the regulation of the protein's function and ciliogenesis, we found that overexpression of either domain interfered with the normal regulation of CEP290 and was sufficient to initiate aberrant primary cilium formation. This demonstrates that there is no further downstream regulation of ciliogenesis beyond CEP290. Overexpression of the regulatory regions in serum-starved cells, where CEP290 is known to be relieved of inhibition, did not result in any increase in CEP290 activity, implying that these regulatory domains act through the same pathway that mediates normal CEP290 inhibition.

These data support a definition for a mechanism of CEP290 regulation. In one case, both regulatory loci could be acted upon by extraneous inhibitory factors to mediate CEP290 inhibition. In fact, it has been shown that the protein CP110 acts as just such an inhibitory factor, binding to the N-terminus of CEP290 and inhibiting protein activity (18, 19). Accordingly, we found that, in some cells, overexpression of CEP290's N-terminus led to the growth of multiple ciliary axonemes. This is consistent with what would be expected upon competition between endogenous CEP290 and the overexpressed N-terminal fragment for CP110, normally removed from only one end of one centriole to initiate ciliogenesis. Nonspecific depletion of CP110 from both ends of both centrioles by the overexpressed N-terminus could have resulted in the growth of multiple ciliary axonemes in the cells we observed.

If inhibition were solely dependent on the binding of a finite pool of endogenous inhibitory factor, then overexpression of the full length protein should result in competition for the inhibitory factor and only minimal, if any, observed inhibition of the overexpressed protein. Additionally, we would not expect to see inhibition of the full length construct in in vitro assays where inhibitory factors should not be present at meaningful concentrations. In all our experiments assessing protein activity, we found that full length CEP290 was significantly inhibited compared to truncation mutants lacking the novel inhibitory regions, arguing against extraneous factors such as CP110 being solely responsible for CEP290 regulation.

Without wishing to be bound by theory, we propose an alternative model for CEP290, i.e., the two novel regulatory regions of the protein cooperate to inhibit CEP290 function, binding to each other and causing a conformational change in the protein, stabilized by the binding of CP110, that obscures important functional domains and decreases protein function (FIG. 8). Thus, the overexpression of either regulatory domain would saturate endogenous CEP290 regulatory domains, preventing the protein from homotypically binding to itself and resulting in a paradoxical increase in protein function. Similarly the full length protein, of its own accord, would be expected to display innate inhibition regardless of the experimental conditions used. These are exactly the results we observed in all of the examples.

CEP290's amphipathic helix falls within the N terminal regulatory region we have identified, positioning it appropriately to play a similar role in the autoinhibition of the protein. Additionally, the microtubule binding region we have identified is immediately adjacent to and potentially a component of the novel C-terminal inhibitory domain. This mechanism of autoinhibition, conserved among membrane-actin cytoskeleton bridging proteins, is likely operative and conserved for membrane to microtubule bridging proteins as well. CEP290 would be the first protein in this class shown to rely upon this mechanism of inhibition.

This model is also supported by observations made in the rd16 mouse, such as the apparent increased affinity of rd16 Cep290, compared to WT, for RPGR. The rd16 deletion of the microtubule binding/regulatory region may thus result in decreased autoinhibition and a higher affinity of Cep290 for its interacting partners, as was observed.

Numerous organ systems are affected by CEP290 deficiencies. The microtubule binding function of CEP290 is clearly of critical importance to the function of the protein on a cellular level and, critical in disease. The rd16 mouse Cep290 gene was found to encode a version of the protein completely deficient in microtubule binding. This mouse model also exhibits significant deficits in primary cilium formation and dramatic and rapid retinal degeneration, implying that deficiencies in microtubule binding can lead to significant pathology. In fact, over 24 unique mutations identified in human CEP290 patients map to the novel microtubule binding domain we report here (FIG. 1). Almost all of these mutations are expected to have truncating effects on the protein, which, as in the rd16 mouse, would result in significant deficiencies in microtubule binding. These truncated effects can explain the mechanism underlying the disease phenotype seen in these individuals.

Example 11: Creation of a CEP290 Knockdown Cell Line

To test the efficacy of our miniCEP290 therapeutic we first set out to create a CEP290-knockdown reporter cell line. Knockdown of CEP290 in cultured cells has been reported to result in dramatic decreases in ciliation upon serum starvation, and rescue of this phenotype serves as a good reporter for miniCEP290 protein function. Three CEP290 shRNA constructs were generated and transfected into hTERT-RPE1 cells to assess their ability to knockdown levels of CEP290 protein. One week post transfection cell lysates were collected and assayed for CEP290 by western blotting. All three constructs were found to affect a greater than 50% reduction in CEP290 protein levels, even in only transient transfection (FIGS. 12A, 12B). To assay the effects of each of these constructs on cell ciliation, retrovirus vectors encoding each construct were generated and used to transduce hTERT-RPE1 cells. Control and transduced cells were stained for acetylated tubulin and observed by fluorescence microscopy for deficits in ciliation. Construct sh2 in particular was found to affect a profound decrease in cell ciliation, with only 50% of cells forming primary cilia, compared to 75% of control cells, upon serum starvation (FIG. 12C, 12D).

We selected CEP290 shRNA construct 2 due to its efficiency in knocking down CEP290 protein levels and ability to affect a significant ciliary phenotype. CEP290 sh2 retrovirus-transduced hERT-RPE1 cells were selected for with puromycin and individual clones were isolated and assayed for CEP290 levels. All of the clones tested were found to exhibit significant knockdown of CEP290 (FIG. 13A), but clones 2.5, 2.7, and 2.8 were each found to affect the greatest knockdown, with less than 30% of endogenous CEP290 remaining (FIG. 13B). To test the ciliary phenotype of such significant knockdown of CEP290 protein, clonal lines 2.5, 2.7, and 2.8 were stained for acetylated tubulin, a marker of the primary cilium, and assayed by fluorescence microscopy for ciliation. Clone 2.8 in particular was characterized by a remarkably severe deficit in ciliation upon serum starvation (FIG. 13C), with only 5% of sh2.8 cells producing primary cilia, compared to greater than 75% of control cells (FIG. 13D). In the serum fed state, sh2.8 cells formed cilia only extremely rarely, making accurate quantification difficult (data not shown). CEP290 shRNA 2.8 cell line was selected as a reporter for miniCEP290 gene rescue.

Example 12—Design of a Minicep290 Gene

To design a miniCEP290 gene small enough to fit within AAV's limited packaging capacity and to maintain as much of the protein as possible, CEP290 amino acids 130-380, 700-1040, 1260-1605, and 1695-1990 were included, making for a total open reading frame of only 3.7 kb, small enough for AAV packaging (FIG. 14A, 14B). MiniCEP290 was thus strategically designed to include CEP290's membrane and microtubule binding domains and almost all of CEP290's PCM-1, NPHP5, CC2D2A, and Rab8A binding domains, while leaving out as much of CEP290's known autoregulatory domains as possible. The mini gene was codon optimized, both to increase expression levels and to harden the transcript against the effects of the shRNA used to generate the CEP290 knockdown reporter cell line, and synthesized by DNA 2.0.

Example 13—Minicep290 Localizes Correctly in HTERT-RPE1 Cells

As a preliminary test of miniCEP290's ability to recapitulate CEP290 functionality, a GFP-fused miniCEP290 expression construct was created. Upon transfection into hTERT-RPE1 cells, miniCEP290 was found to localize to discrete puncta throughout the cytoplasm (FIG. 14C), a pattern similar to that observed for overexpressed full-length CEP290. In cells where primary cilia were present, a punctus of miniCEP290 was always found exactly at the ciliary transition zone, immediately between the ciliary axoneme and centrosome (as indicated by acetylated tubulin and pericentrin staining, respectively; FIG. 14C), indicating that, at least in cells with endogenous CEP290 expression, miniCEP290 was capable of correctly localizing to the same compartment as its full-length counterpart.

MiniCEP290 localizes appropriately in hTERT-RPE1 cells and doesn't seem to interfere with ciliogenesis, an important concern when overexpressing truncation mutants. To determine whether miniCEP290 is also capable of binding to microtubules and cellular membranes, two functions recently discovered to be critical to full-length CEP290's role in cilium formation, a panel of immunoprecipitation experiments is performed to confirm that the minigene is capable of interacting with known CEP290 binding partners, an aspect of CEP290 biology important to the protein's function at the cilium. The miniCEP290 gene is subcloned into a lentiviral vector to assess its ability to rescue the ciliogenesis phenotype of the sh2.8 cell line.

Results in cell culture are validated in animal models to demonstrate therapeutic use. The lentiviral vectors are tested in early postnatal rd16 mice as this leads to transduction of at least some photoreceptor precursor cells. Also, AAV vectors encoding the miniCEP290 gene are generated and subretinally administered to rd16 mice. Since miniCEP290 recapitulates some of full-length CEP290's function, we anticipate at least partial correction of the rd16 retinal degeneration phenotype in the treated eyes.

Example 14: In Vivo Studies in a CPE290 Mutant Animal

For in vivo studies, virus is delivered subretinally or intravitreally under direct surgical visualization using methods described previously (Bennett, J., et al. 1999 Proc. Natl. Acad. Sci. USA 96, 9920-9925 and Bennett, J. et al, 2000 Meth. Enzymol. 316, 777 to 789). Eyes from three animals, e.g., mice or dogs, are injected either subretinally or intravitreally with an AAV8bCEP290 vector or AAV2/5 CEP290 vector, each containing the minigene CEP290 of SEQ ID NO: 5 under control of the rhodopsin kinase promoter. Control eyes are untreated. Other animals are kept as untreated controls.

The compositions containing the AAV vectors in isotonic sodium chloride solution are administered by subretinal injection at suitable concentrations—for larger animals about 1.5×10¹¹ vg/ml in volumes of 150 to 400 μl and in smaller concentrations or volumes for mice. Any detachments are anticipated to resolve spontaneously within 24 hours. Animals are evaluated post-operatively for evidence of ocular or systemic toxicity, virus exposure to extraocular tissue, virus shedding, unfavorable immune response or other untoward effects. None is expected to be found.

Eyes are evaluated clinically at regular intervals following the surgery to identify inflammation. Humoral and intraocular antibodies specific to AAV capsid proteins are evaluated as described in Bennett, J., et al. 1999 Proc. Natl. Acad. Sci. USA 96, 9920 to 9925, incorporated herein by reference. Hematology and blood chemistries are expected to reveal no evidence of systemic toxicity.

To correlate transgene expression with changed visual function, one subretinally injected eye is surgically enucleated 99 days post injection. The eyecup is divided into temporal-superior, temporal-inferior, nasal-superior, and nasal-inferior quadrants. From each quadrant, the retina, and photoreceptor cells are separately harvested and dissected under RNase free conditions and rapidly frozen. Total RNA is prepared from these cells using the TRIzol Reagent kit (Life Technologies, Gaithersburg, Md.). DNA is extracted from the same tissues according to the vendor's protocol. cDNA is amplified from total RNA using RNA PCR kit (Perkin Elmer, Foster City, Calif.) and the conditions listed above.

Genomic PCR demonstrates persistence of transferred viral DNA photoreceptor cells. From noninfected cells of the affected animal, only mutant product amplifies, but several days posttransfection in vitro the CEP290 minigene should yield the overwhelming product.

RT-PCR (figures not shown) demonstrates expression of CEP290 minigene message in photoreceptor cells. PCR analyses of serum and tear fluid show no sign of virus shedding after injection. Reverse transcriptase (RT)-PCR on sera, conjunctiva, eyelids, the gland of the third eyelid, and the optic nerve from the enucleated eye of BR29 are anticipated to be negative for the transgene many days post injection, indicating that virus escape to extraocular tissues is below detectable levels.

Retinal and visual function testing is then conducted using electroretinograms (ERGS). The physiological consequences of the treatments are assessed by electroretinography (ERG) (Banin, E., et al. 1999 Neuron 23, 549 to 57). Animals are dark-adapted (overnight), premedicated with acepromazine (0.55 mg/kg, IM) and atropine (0.03 mg/kg, IM) and anesthetized by intermittent ketamine (15 mg/kg, IV, repeated every 15 minutes). Pulse rate, oxygen saturation and temperature are monitored throughout. The cornea is anesthetized with topical proparacaine HCl (1%) and pupils dilated with cyclopenylate (1%) and phenylephrine (2.5%).

Full field ERGs are recorded using a computer-based system (EPIC-XL, LKC Technologies, Inc., Gaithersburg, Md.) and Burian-Allen contact lens electrodes (Hansen Ophthalmics, Iowa City, Iowa) (Banin, E., et al. 1999 Neuron 23, 549-57). Dark-adapted luminance-response functions are obtained with blue (Wratten 47A) flash stimuli spanning ˜6 log units (−2.9 to +2.8 log scottocd·s·m⁻²).

ERG b-wave amplitudes are measured conventionally from baseline or a-wave trough to positive peak; a-wave amplitude is measured from baseline to negative peak at the maximal stimulus. For isolating cone pathway function, animals are light-adapted and 29 Hz flicker ERGs evoked with white flash stimuli (0.4 log cd·s·m⁻²) on a background (0.8 log cd·m⁻²); amplitudes are measured between successive negative and positive peaks and timing from stimulus to the next positive peak. Ocular axial length and pupil diameter are measured for each experiment to permit calculation of retinal illuminance.

The restoration of retinal/visual function in the experimental model by subretinal AAV to RPE65 is demonstrated by the results of the above-described ERGs. A comparison of dark-adapted ERGs evoked by increasing intensities of blue light stimuli in a control animal with ERGs to the same stimuli in the disease model animal shows the affected animal has elevated thresholds, reduced amplitudes and waveform shape changes (i.e., b-waves but no detectable a-waves). Over a 5 log unit range of increasing stimulus intensity, the ERG of normal animals are anticipated to respond with increasing amplitude of bipolar cell (b-wave) and photoreceptor (a-wave) components. At all intensities these signals are dominated by rod photoreceptor retinal pathways. Compared to normal animals, the threshold stimulus required to elicit an ERG response from model animals is elevated.

Retinal function is improved in eyes treated with subretinal AAV to CEP290, compared to pretreatment recordings. After subretinal AAV to RPE65 therapy, the mutant animal is anticipated to show an improved b-wave threshold, a large increase of a- and b-wave amplitudes (although not to normal levels) and an ERG waveform shape that is similar to controls.

The details of photoreceptor function are analyzed by the amplitude and timing of the ERG photoresponses evoked by 2.8 log scottocd·s·m⁻² flashes. All eyes with subretinal AAV-CEP290 fragment treatment recover cone flicker responses. Cone flicker ERGs are readily recordable post-treatment).

Transmission of retinal activity to higher visual pathways is demonstrated by pupillometry. Animals are dark-adapted for more than 3 hours and pupil responses are obtained sequentially from each eye using full-field green stimuli (−3.2 to +3.0 log scot-cd·m⁻²) of −2 second duration. Pupils are imaged with a video camera under infrared illumination and continuously recorded on a VCR. Dynamic changes in pupil diameters are measured from single frames displayed on the video monitor in relation to the timing of each stimulus. Pupil responses are calculated by subtracting the smallest pupil diameter achieved within 2 seconds after the stimulus onset from the diameter measured in the dark.

All tested pupils are expected to constrict in response to high intensity stimuli. The threshold intensity to reach a criterion pupillary response is anticipated to be improved in subretinally-treated eyes compared with untreated eyes.

Qualitative visual assessment of treated animals is performed at post injection using an obstacle course and observers masked to the experimental design. Visual behavior is also documented by video recording. Results of behavioral testing are anticipated to be consistent with the electrophysiological results.

Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and illustrative examples, make and utilize the compositions of the present invention and practice the claimed methods. While the invention has been described and illustrated herein by references to various specific materials, procedures and examples, it is understood that the invention is not restricted to the particular combinations of material and procedures selected for that purpose. The disclosures of T. G. Drivas et al, J Clin Invest, 2013; 123(10); 4525-4539 and U.S. provisional patent application No. 61/847,016, as well as all patents, patent applications and other references, including the Sequence Listing cited in this specification are hereby incorporated by reference in their entirety.

TABLE 2 (Sequence Listing Free Text) The following information is provided for sequences containing free text under numeric identifier <223>. SEQ ID NO: (containing free text) Free text under <223> 3 Synthetic construct having codon optimization of human nucleic acid sequence for CEP290 4 Synthetic construct having codon optimization of human protein sequence for CEP290 5 Synthetic Construct; nucleic acid sequence containing codon optimized human CEP290 fragments spliced together in a single open reading frame; the nucleic acid sequences encoding fragments aa130to380, aa700to1040; aa1260to1605 and aa1695to1990. 6 Synthetic Construct; amino acid sequence containing codon optimized human CEP290 fragments spliced together in a single open reading frame: aa130to380, aa700to1040; aa1260to1605 and aa1695to1990.

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1. A method of treating a mammalian subject having a disease associated with a defect in the CEP290 gene, a CEP290 mutation, or a defect in the expression or levels of expression of the CEP290 protein, the method comprising administering to said subject an effective concentration of a composition comprising: (i) a synthetic or recombinant nucleic acid sequence encoding a truncated CEP290 protein comprising discontinuous CEP290 fragments spliced together in a single open reading frame, said truncated CEP290 protein having biological activity that mimics the biological activity of normal full-length CEP290, wherein the truncated CEP290 protein comprises the amino acid sequence of SEQ ID NO: 6; and (ii) a therapeutically acceptable or pharmaceutically acceptable carrier.
 2. The method of claim 1, wherein the administering comprises delivering the composition by subretinal injection to the retina or photoreceptor cells, delivering the composition by retrograde urethral injection to the kidney, delivering the composition by intra-ventricular or intra-cerebral injection to the brain, or topically delivering the composition to nasal epithelium.
 3. The method of claim 1, wherein the mammalian subject is a human.
 4. A method of treating a mammalian subject having a disease associated with a defect in the CEP290 gene, a CEP290 mutation, or a defect in the expression or levels of expression of the CEP290 protein, the method comprising administering to said subject an effective concentration of a composition comprising: (i) a synthetic or recombinant nucleic acid sequence encoding a truncated CEP290 protein comprising discontinuous CEP290 fragments spliced together in a single open reading frame, said truncated CEP290 protein having biological activity that mimics the biological activity of normal full-length CEP290, wherein the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 5; and (ii) a therapeutically acceptable or pharmaceutically acceptable carrier.
 5. The method of claim 4, wherein the administering comprises delivering the composition by subretinal injection to the retina or photoreceptor cells, delivering the composition by retrograde urethral injection to the kidney, delivering the composition by intra-ventricular or intra-cerebral injection to the brain, or topically delivering the composition to nasal epithelium.
 6. The method of claim 4, wherein the mammalian subject is a human.
 7. A method of treating a mammalian subject having a disease associated with a defect in the CEP290 gene, a CEP290 mutation, or a defect in the expression or levels of expression of the CEP290 protein, the method comprising administering to said subject an effective concentration of a composition comprising: (i) a synthetic or recombinant nucleic acid sequence encoding a truncated CEP290 protein comprising discontinuous CEP290 fragments spliced together in a single open reading frame, said truncated CEP290 protein having biological activity that mimics the biological activity of normal full-length CEP290, wherein the discontinuous CEP290 fragments comprise the amino acid sequence of SEQ ID NO: 49 and the amino acid sequence of SEQ ID NO: 53; and (ii) a therapeutically acceptable or pharmaceutically acceptable carrier.
 8. The method of claim 7, wherein the administering comprises delivering the composition by subretinal injection to the retina or photoreceptor cells.
 9. The method of claim 7, wherein the administering comprises delivering the composition by retrograde urethral injection to the kidney.
 10. The method of claim 7, wherein the administering comprises delivering the composition by intra-ventricular or intra-cerebral injection to the brain.
 11. The method of claim 7, wherein the administering comprises topically delivering the composition to nasal epithelium.
 12. The method of claim 7, wherein the discontinuous CEP290 fragments further comprise the amino acid sequence of SEQ ID NO:
 51. 13. The method of claim 7, wherein the discontinuous CEP290 fragments further comprise the amino acid sequence of SEQ ID NO:
 52. 14. The method of claim 7, wherein the discontinuous CEP290 fragments further comprise the amino acid sequence of SEQ ID NO: 51 and the amino acid sequence of SEQ ID NO:
 52. 15. The method of claim 7, wherein the mammalian subject is a human.
 16. The method of claim 7, wherein the mammalian subject has shown a clinical sign of ciliopathy.
 17. The method of claim 7, wherein the mammalian subject has Lebers Congenital Amaurosis (LCA).
 18. The method of claim 16, wherein the clinical sign comprises decreased peripheral vision, decreased central vision, decreased night vision, loss of color perception, reduction in visual acuity, decreased photoreceptor function, or a pigmentary change.
 19. The method of claim 16, wherein the ciliopathy is a disorder of the bone, brain, central nervous system, kidney, or olfactory epithelia. 